|
Figure 2. Spatial and temporal analysis of Xdhcr7 expression. A: WMISH analysis of the expression of Xdhcr7 and Xshh. Staging according to Nieuwkoop and Farber (1975). a-i: WMISH analysis of Xdhcr7 expression. e', h': Transversal sections as indicated by black dashes in e and h, respectively. a-d: Vegetal view (dorsal to the top) of gastrula stage embryos showing the expression of Xdhcr7 in the dorsal blastopore lip (a, b), and in the forming dorsal midline (c, d). e,f: Dorsal view (anterior to the bottom) of neural plate (e) and early neural tube (f) stage embryos. Xdhcr7 highly stains the dorsal midline (e), and additional staining appears in the epidermis (ep) at neural tube stage (f). e': The expression of Xdhcr7 in the notochord. g, h: Lateral view (anterior to the left) of late neural tube (g) and early tailbud (h) stage embryos. Xdhcr7 is expressed in the notochord, the neural tube, the optic vesicle, the head epidermis, and the epibranchial placodes. Red arrow indicates the posterior limit of the Xdhcr7 expression domain in the neural tube. h': The expression of Xdhcr7 in the hypochord, the notochord, and the lateral ventral neural tube, but not in the floor plate (inset). i: Lateral view (anterior to the left) of the head region of a late tailbud stage embryo; Xdhcr7 expression is restricted rostrally, and mainly demarcates neural structures. j,k': WMISH analysis of Xshh expression. j', k': Transversal sections as indicated by black dashes in j and k, respectively. j: Dorsal view (anterior to the bottom) of a neural plate stage embryo. Xshh is expressed in the dorsal midline and the prechordal plate. j': The expression in the notochord. k: Lateral view (anterior to the left) of a tailbud stage embryo showing the expression of Xshh. k': The expression of Xshh in the notochord and floor plate, but not in the lateral ventral neural tube. l: Schematic illustration of the spatial distribution of Xdhcr7 and Xshh transcripts at tailbud stage. Xdhcr7 and Xshh transcripts colocalise only in the notochord and hypochord, but are expressed in neighbouring domains in the neural tube. B: RT-PCR analysis of the appearance of Xdhcr7 and Xshh transcripts during development and in adult tissues. RT-PCR primers were designed to amplify nucleotides 431 to 805 of Xdhcr7. Zygotic expression of Xdhcr7 starts at around stages 10 to 11, which coincides with the start of Xshh expression. Transcript levels of Xdhcr7 drop at around tailbud stage (st. 32), when Xshh is still highly expressed. The relative distribution of Xdhcr7 and Xshh mRNA in adult tissues is quite similar, as they are both highly expressed in the brain, eye, spinal cord, skin, kidney, lung, liver, stomach, and testis. Histone H4 was probed as RNA loading control. C: WMISH analysis of the expression of Xshh (a, d), Xsal-1 (b, e), and Xdhcr7 (c, f) in banded hedgehog (Xbhh) injected embryos. Xbhh capped RNA (500 pg) was injected (in combination with 200 pg of beta -galactosidase capped RNA as linage tracer) into one blastomere of two-cell-stage pigmented embryos. a-c: Transversal sections at the level of the eyes of stage 34 embryos. d-f: Transversal sections at the level of the trunk of stage 34 embryos. a, d: Expansion of Xshh expression in the forebrain, but not in the floor plate and notochord. b, e: Expansion of Xsal-1 expression in the brain and neural tube. c, f: Xbhh suppresses the expression of Xdhcr7 in neural structures (eye, brain, and spinal chord), but not in the notochord (c'). br, brain; cs, control side; dbl, dorsal blastopore lip; dml, dorsal midline; ebp, epibranchial placodes; ep, epidermis; ey, eye; fb, fore brain; fp, floor plate; hb, hind brain; hc, hypochord; is, injected side; lvnt, lateral ventral neural tube; mb, midbrain; nc, notochord; nt, neural tube; ot, otic vesicle; ov, optic vesicle; pp, prechordal plate; Sp. cord, spinal cord.
|
|
Figure 4. Mevinolin treatment and Dhcr7-knockdown cause comparable phenotypes. a-d', i-i rdquo : Untreated embryos. e-h', j-j rdquo : Embryos treated with 125 mu M Mevinolin. k-k rdquo : Embryo treated with 25 mu M AY9944. l-l rdquo : Embryo treated with 250 mu M AY9944. m-m rdquo : Embryo treated with a combination of 25 mu M AY9944 and 12.5 mu M Mevinolin. The embryos are all treated from stage 8. a, c, e, g: Anterior views (dorsal up). b', d', f', h': Frontal views (dorsal up). b, d, f, h, i, i', j, j', k, k', l, l', m, m': Lateral views (dorsal up), (i rdquo , j rdquo , k rdquo , l rdquo , m rdquo ) dorsal views. a-d', e-h': WMISH analysis of the expression of Xsix3 (a-b'; e-f') and Xrx1/Xkrox20/Xen2 (c-d'; g-h'). j-m rdquo : Head phenotype. e-h': Early Xsix3 (e) and Xrx1 (g) expression is not affected by Mevinolin treatment; however, their expression at tailbud stage (f-f', h-h') is severely reduced. j-j': At tadpole stage, the Mevinolin-treated embryo displays small eyes that are fused to the midline, with a failure of the optic cup to close properly. k-k rdquo : Treatment with 25 mu M AY9944 does not cause any obvious phenotype. l-l rdquo : AY9944 treatment at 250 mu M results in developmental delay, small head phenotype with a loss of cement gland (arrow in l), and bended tail. The eyes are small, shifted towards the midline, and display a colobama. m-m rdquo : Combination of 25 mu M AY9944 and a low concentration of Mevinolin results in the same phenotype as a high concentration of AY9944.
|
|
Figure 3. Impairment of eye development upon Xdhcr7 knockdown. a-j rdquo : Microinjection of Xdhcr7-morpholino (1 pmol) in one blastomere of two-cell-stage embryos. In combination, 200 pg of beta -galactosidase mRNA was injected as linage tracer (blue staining). k-k rdquo ': Microinjection of Xdhcr7-morpholino (0.5 pmol) in two blastomeres of two-cell-stage embryos. l: Microinjection of Xdhcr7-morpholino (1 pmol) in two blastomeres of two-cell-stage embryos (a, c, e) Anterior view (dorsal up); dashed lines indicate the midline. b, b rdquo , d, d rdquo , f, f rdquo : Lateral view (dorsal up). k rdquo : Ventral view (anterior down). b', d', f': Frontal view (dorsal up). g-l: Dorsal view (anterior down). f rdquo ', g', h', j', j rdquo , k rdquo ': Transversal sections. b rdquo ', d rdquo ': Horizontal sections; dashed lines indicate the midline. a-h': WMISH analysis of Xrx1/Xen2/Xkrox20 (a-b rdquo '), Xsix3 (c-d rdquo '), Xn-tubulin (e-f rdquo '), Xgsh1 (g, g'), and Xnkx2.2 (h, h') expression. i-l: Eye phenotypes. a: Suppression of Xkrox20 (r3 and r5; 40%, n = 25) and Xen2 (mhb; 40%, n = 25) expression. a,c: Expansion of Xrx1 (48%, n = 25) and Xsix3 (56%, n = 25) expression at neurula stage. b-b rdquo ': Reduction of Xrx1 (56%, n = 32), Xen2 (53%, n = 32), and Xkrox20 (65%, n = 32) expression at tailbud stage. The expression domain of Xrx1 is shifted towards the midline (b rdquo '). d-d rdquo ': Severe reduction of Xsix3 expression (67%, n = 28). Retinal Xsix3 expression is almost linked to the brain (d rdquo '). e: Severe reduction of Xn-tubulin expression at neurula stage (72%, n = 29). Note the reduced expression in both the neural plate and the trigeminal placode. f-f rdquo ': Severe reduction of Xn-tubulin expression at tailbud stage (57%, n = 35). Xn-tubulin expression is almost absent in the neural tube (compare injected and non-injected side in f rdquo '). g-h': Suppression of Xgsh1 (63%, n = 30) and Xnkx2.2 (59%, n = 32) expression. The expression domain of Xnkx2.2 is slightly shifted ventrally. i-i rdquo : Small eye (60%, n = 300). The small eye is closer to the brain and displays a coloboma (i rdquo ). j-j rdquo : Severely reduced eye formation (33%, n = 300). The retinal pigmented epithelium is almost absent in the eye of the injected side (white arrowhead in j rdquo ). k-k rdquo ': Mild eyes phenotype induced by injection of 0.5 pmol Xdhcr7-morpholino in two cells of two-cell stage. l: Cyclopic eye induced by bilateral injection of 1 pmol Xdhcr7-morpholino. cp, cranial placode; cs, control side; ey, eye; is, injected side; le, lens; mhb, midbrain-hindbrain boundary; r3, rhombomere 3; r5, rhombomere 5; rpe, retinal pigmented epithelium; tp, trigeminal placode.
|
|
Figure 5. Impairment of eye development upon Dhcr7 overexpression in Xenopus. a-j: Microinjection of mouse Dhcr7 (mDhcr7, 2 ng) into one blastomere of two-cell-stage embryos. In combination, 200 pg of beta -galactosidase mRNA was injected as linage tracer (blue staining). a, c, e: Anterior view (dorsal up). b, b rdquo , d, d rdquo , f, f rdquo : Lateral view (dorsal up). b', d', f': Frontal view (dorsal up). g, h, i, j: Dorsal view (anterior down). b rdquo ', d rdquo ': Horizontal sections. f rdquo ', g', h', i', i rdquo : Transversal sections. a-h': WMISH analysis of Xrx1/Xen2/Xkrox20 (a-b rdquo lsquo ), Xsix3 (c-d rdquo '), Xn-tubulin (e-f rdquo rsquo ), Xgsh1 (g, g'), and Xnkx2.2 (h, h') expression. i-j: Eye phenotype. a, c: Early expression of Xrx1 (e) and Xsix3 is mildly expanded (a and c, compare the injected and non-injected sides). The expression of Xkrox20 (r3 and r5) and Xen2 (mhb) show a slight reduction, especially in the 5th rhombomere (Xkrox20). b, b', b rdquo , d, d', d rdquo : Reduced expression of Xrx1 (b-b rdquo ; 43%, n = 30) and Xsix3 (d-d rdquo ; 60%, n = 30) at tailbud stage. e: Suppression of the early expression of Xn-tubulin in the trigeminal placode (36%, n = 25). f-f rdquo ': Reduction of Xn-tubulin staining in the cranial placodes at tailbud stage (48%, n = 40; red arrowheads in f rdquo ). f rdquo ': Expansion of the neural tube on the injected side (dashes). g, g': Expansion of Xgsh1 expression (47%, n = 32). h, h': Slight expansion of Xnkx2.2 expression (33%, n = 30). i, j: Small eyes (55%, n = 200) and/or pigmented optic stalks (30%, n = 200; red arrowhead in j) induced by mDhcr7 injection. The overall patterning of the injected side's retina is not affected (i' and i rdquo ). cp, cranial placode; cs, control side; is, injected side; le, lens; mhb, midbrain-hindbrain boundary; r3, rhombomere 3; r5, rhombomere 5; rpe, retinal pigmented epithelium; tp, trigeminal placode.
|
|
Figure 6. The alternative spliced isoforms of Xdhcr7 differentially affect eye development upon overexpression. a-d rdquo , k: Microinjection of Xdhcr7-M (2 ng) into one blastomere of two-cell-stage embryos. e-h rdquo , i-j rdquo , l-l rdquo : Microinjection of Xdhcr7-S (750 pg) into one blastomere of two-cell-stage embryos. In combination, 200 pg of beta -galactosidase mRNA was injected as linage tracer (blue staining). a, c, e, g: Anterior view (dorsal up); dashes indicate the midline. b, b rdquo , d, d rdquo , f, f rdquo , h, h rdquo : Lateral view (dorsal up). b', d', f', h': Frontal view (dorsal up). i, j, k, l: Dorsal view (anterior down). i', j', l'-l rdquo : Transversal sections. a-j': WMISH analysis of Xrx1/Xen2/Xkrox20 (a-b rdquo , e-f rdquo ), Xsix3 (c-d rdquo , g-h rdquo ), Xgsh1 (i-i') and Xnkx2.2 (j-j') expression. k-l rdquo : Eye phenotype. a-b rdquo , c-d rdquo : The expression of Xrx1/Xkrox20/Xen2 as well as Xsix3 is not affected upon overexpression of Xdhcr7-M. e: Xdhcr7-S does not affect the early expression of Xrx1, Xkro20 (r3 and r5), and Xen2 (mhb), although the late expression of Xrx1 is reduced (f-f rdquo , 30%, n =3 0). g: Slight expansion of the early expression of Xsix3 upon overexpression of Xdhcr7-S; however, the late expression (h-h rdquo ) is severely reduced (62%, n = 26). i-j': Xdhcr7-S causes a reduction of the expression of Xgsh1 (i-i'), but not obviously Xnkx2.2 (j-j'). k: No obvious alteration of eye formation is induced by Xdhcr7-M overexpression. l: Small eye phenotype induced by Xdhcr7-S (38%, n = 200). l'-l rdquo : The retinal patterning of the Xdhcr7-S-injected eye is not severely affected. cs, control side; is, injected side; le, lens; mhb, midbrain-hindbrain boundary; r3, rhombomere 3; r5, rhombomere 5; rpe, retinal pigmented epithelium.
|
|
Fig. 7. Organisation of retinal cell layers upon manipulation of the Xdhcr7 activity. a: Paraffin sections (5 m) of control and injected embryos. a: Section of control embryo. b: Section of Xdhcr7-morpholino injected embryo. c: Section of mDhcr7-injected embryo. d: Section of Xdhcr7-S-injected embryo. a: Phase contrast images. a d Fluorescent images of DAPI- stained sections. ad Fluorescent images of TR-WGA-stained sections. ad: Overlays of DAPI and TR-WGA staining. a: From proximal to distal, different retinal cell layers can be clearly distinguished, comprising the ganglion cell layer (cell bodies of ganglion and amacrine cells), the inner plexiform layer (synaptic connections between bipolar, amacrine, and ganglion cells), the inner nuclear layer (cell bodies of horizontal, bipolar, and amacrine cells), the outer plexiform layer (dendritic processes of bipolar and horizontal cells), and the outer nuclear layer (containing photoreceptors). a a: DAPI stains the nuclei and demarcates three layers in the retina (gcl, inl, onl). a a: TR-WGA strongly decorates rods and cones (ph) matrix domain and weakly stains the inner plexiform layer (ipl). b: The eye of Xdhcr7-morpholino-injected embryo is almost linked to the brain. The retinal pigmented epithelium layer is severely reduced (red dashes). The stratification of the retina is only rudimentary, since retinal layers could not be defined (b b). c: The eye on the mDhcr7-injected side does not show any apparent disorganisation of retinal stratification. d: Xdhcr7-S has a minor effect on the organisation of retinal cell layers. The separation of retinal layers is less sharp than in the control eye, but the boundaries are distinguishable. br, brain; DAPI, 4,6-Diamino-2-phenylindol; gcl, ganglion cell layer; inl, inner nuclear layer; ipl, inner plexiform layer; le, lens; onl, outer nuclear layer; opl, outer plexiform layer; PC, phase contrast; ph, photoreceptors; rpe, retinal pigmented epithelium; TR-WGA, Tetramethyl-rhodamin-conjugated wheat-germ agglutinin. Scale bars 100 m(a, c, d), 50 m (bd).
|
|
dhcr7 (7-dehydrocholesterol reductase ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, vegetal view, dorsal up.
|
|
dhcr7 (7-dehydrocholesterol reductase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, anterio-dorsal view, dorso-posterior up.
|
|
dhcr7 (7-dehydrocholesterol reductase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left dorsal up.
|
|
dhcr7 (7-dehydrocholesterol reductase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior left dorsal up.
|
|
six3 (SIX homeobox 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, anterior view, dorsal up.
|
|
|
|
Section of control embryo. From proximal to distal, different retinal cell layers can be clearly distinguished: the inner nuclear layer (inl), outer nuclear layer (onl), inner plexiform layer ( ipl), outer plexiform layer (opl), ganglionic cell layer ( gcl), lens ( le) and photoreceptor layer (ph).
|
