QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 12, 2007
doi: 10.1242/10.1242/dev.02885

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Barembaum, M. Articles by Bronner-Fraser, M. Search for Related Content PubMed PubMed Citation Articles by Barembaum, M. Articles by Bronner-Fraser, M.

Spalt4 mediates invagination and otic placode gene expression in cranial ectoderm

Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA.

View larger version (108K): [in this window] [in a new window]   Fig. 1. Expression of Spalt4 in ectoderm and derived tissues. Whole-mount in situ hybridization of (A) stage 3 chicken embryo, (B) stage 5 chicken embryo (arrows indicate level of sections in D and E), (C) stage 8 chicken embryo (arrow indicates levels of sections in F). (D) Section through embryo in (B) at the level of the notochord. (E) Section through embryo in B at the level of Hensen's node. (F) Section through stage 8 embryo at midbrain level. (G) Expression of Spalt4 in a stage 10 embryo sectioned at hindbrain level. (H) Expression of Spalt4 in the otic pit (opi) of a stage 13 embryo. Arrowhead points to the neural crest. (I) Expression of Spalt4 in the otic vesicle (otv) of a stage 16 embryo. Arrowhead indicates the neural crest. (J) Section through a stage 14 embryo at eye level. (K) Section through a stage 17 embryo at forebrain level. (L) Whole-mount in situ hybridization with Spalt4 in a stage 19 embryo showing strong Spalt4 expression in the forelimb. Arrow shows the level of the section in M. (M) Section through the forelimb in L. (N-Q) Comparison of stage 5 expression of (N). Spalt4 (O). Six1 (P). Eya2 and (Q) Sox3. ps, primitive streak; hn, Hensen's node; no, notochord; otp, otic placode; opi, otic pit; otv, otic vesicle; mb, midbrain; hb, hindbrain; re, retina; le, lens; fb, forebrain; ol, olfactory epithelium; fl, forelimb; d, dorsal; v, ventral; aer, apical ectodermal ridge.

View larger version (51K): [in this window] [in a new window]   Fig. 2. Fgf2-soaked bead induces Spalt4. (A) Whole-mount in situ hybridization 5 hours after Fgf bead implantation. Arrow points to the bead. (B) Higher magnification of bead in (A). Arrow shows plane of section in C. (C) Section of region around bead in B. Arrowhead points to Spalt4 signal. (D) Whole-mount in situ hybridization five hours after BSA bead implantation. Arrow points to bead. (E) Higher magnification of region around bead in D. Arrow shows plane of section in F. (F) Section of region around bead in E.

View larger version (74K): [in this window] [in a new window]   Fig. 3. Electroporation of a plasmid driving Spalt4 expression induces the expression of ectopic vesicles. (A) Embryo electroporated with a control GFP plasmid. (B) Embryo in A hybridized with Sox10 RNA probe. (C) Magnified view of embryo in A. (D) Sox10 in situ hybridization of control embryo in C. (E) Embryo electroporated with Spalt4-GFP construct. A number of ectopic vesicles are visible (arrows). (F) The ectopic vesicles in the Spalt4 electroporated embryo in E express Sox10 (arrows). (G) Embryo electroporated with Spalt4-GFP construct. A number of ectopic vesicles are visible (arrows). (H) The ectopic vesicles in the Spalt4 electroporated embryo in G express Notch1 (arrows). (I) Embryo electroporated with Spalt4-GFP construct. A number of ectopic vesicles are visible (arrows). (J) The ectopic vesicles in the Spalt4 electroporated embryo in (I) express EphA4 (arrows). OtV, otic vesicle; BA, branchial arch.

View larger version (132K): [in this window] [in a new window]   Fig. 4. Expression of otic expressed genes in the ectopic vesicles at the level of the hindbrain. (A) Spalt4, (B) Sox10, (C) Notch1, (D) Lunatic fringe, (E) EphA4, (F) Tbx1, (G) Dlx5. (H) Six1 mRNA was not detected in an ectopic vesicle by in situ hybridization (arrow), though surrounding cells were positive. (I) GFP. (J) An antibody to Spalt4. The same section as in I. (K) GFP. (L) Same section as in K showing Pax2 expression. (M) GFP. (N) The same section as in M showing Dlx3 expression. (O) An ectopic vesicle in an embryo 72 hours post electroporation, with the Spalt4 expression construct expressing GFP (green) but there is little detectable TUJ1 staining (red). A portion of an adjacent ganglion shows strong TUJ1 staining. A-F show section in situ hybridizations. G and H are 20 µm cryostat sections of whole-mount in situ hybridizations. The embryos In I-O were paraformaldehyde fixed 48 hours (I-N) or 72 hours (O) after electroporation then sectioned at 10 µm on a cryostat and used for immunohistochemistry. Scale bars: 50 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Misexpression of Spalt4 in otic placodes can prevent the formation of otic vesicles. (A) An embryo electroporated with a control plasmid gives rise to a normal otic vesicle (ov) expressing Sox10. (B) An embryo electroporated with a Spalt4-expressing plasmid gave rise to a flat ectoderm (oe) expressing Sox10 where the otic vesicle is normally located. (C) Section through an embryo electroporated with a control plasmid at the otic vesicle level shows GFP expression in the closed otic vesicle. (D) Same section as in C showing expression of Sox10 in the lateral half of the otic vesicle. (E) Section through an embryo electroporated with a Spalt4-expressing plasmid at otic vesicle level shows GFP-positive cells in the thickened epithelium where the otic vesicle would normally be located. (F) In situ hybridization with Sox10 RNA probe. Same section as in E. (G) Section through an embryo electroporated with a Spalt4-expressing plasmid at otic vesicle level shows GFP expression in the thickened epithelium where the otic vesicle would normally be located. (H) Same section as in G using a Pax2 antibody. (I) Section through otic vesicles of an embryo electroporated with a control plasmid. (J) Same section as in (I) showing Six1 expression in the ventral half of the otic vesicle. (K) Section through the otic ectoderm of an embryo electroporated with a plasmid overexpressing Spalt4. (L) Six1 expression in the same section as in (K). ov, otic vesicle; oe, otic ectoderm; hb, hindbrain.

View larger version (109K): [in this window] [in a new window]   Fig. 6. The effect of expression of Spalt4 in the trigeminal placode. Whole-mount in situ hybridization with a probe to NeuroD in embryos electroporated with (A) control plasmid or (B) plasmid overexpressing Spalt4. Arrows point to the approximate plane of section of trigeminal ganglia in C and D. (C,D) Sections at trigeminal ganglion level of double in situ hybridizations with Sox10 (light blue) and NeuroD (dark blue) in embryos electroporated with (C) control plasmid or (D) plasmid overexpressing Spalt4. (E,F) Sections through an embryo electroporated with Spalt4 on the right side only, and analyzed with GFP (E), or HuD (F) antibody. Scale bars: 100 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 7. Expression of a dominant-negative Spalt4 constructs in the otic placode reduces the size of the otic vesicle. (A) Whole mount and (B) section of an embryo electroporated with the control plasmid and hybridized in situ with a Sox10 RNA probe. (C) Whole mount and (D) section of an embryo electroporated with the dominant-negative Spalt4 plasmid and hybridized in situ with a Sox10 RNA probe. (E) Embryo 8 hours post-electroporation with control plasmid and analyzed with GFP. (F) Same section as in E analyzed for cell death with TUNEL. (G) Embryo 8 hours post-electroporation with dominant-negative Spalt4 plasmid and analyzed with GFP. (H) Same section as in G analyzed for cell death with TUNEL. Scale bars: 200 µm (A,C) 100 µm (B,D).

View larger version (84K): [in this window] [in a new window]   Fig. 8. Mis-expression of Sox10 generates ectopic vesicle that express otic vesicle genes. (A) GFP fluorescence in an embryo electroporated with a control-GFP vector. (B) Embryo in A hybridized with Notch1. (C) An embryo electroporated with a Sox10-GFP construct shows GFP-positive ectopic vesicles (arrows). (D) Embryo in C hybridized with a Notch1 probe. The ectopic vesicles (arrows) express Notch1. (E) An embryo electroporated with a Sox10-GFP construct shows GFP-positive ectopic vesicles (arrows). (F) Embryo in E hybridized with an EphA4 probe. The ectopic vesicles (arrows) express EphA4. (G) An embryo electroporated with a Sox10-GFP construct shows GFP-positive ectopic vesicles (arrows). (H) Embryo in G hybridized with a Spalt4 probe. The ectopic vesicles (arrows) express Spalt4.