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

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 Maves, L. Articles by Kimmel, C. B. Search for Related Content PubMed PubMed Citation Articles by Maves, L. Articles by Kimmel, C. B.

FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain

Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254, USA

View larger version (113K): [in a new window]   Fig. 1. Rhombomere 4 differentiates earliest in the zebrafish hindbrain. (A-D) Single optical sections from a confocal time-lapse recording showing early rhombomere boundary formation. Images show dorsal views with anterior towards the left. Rhombomere boundaries are marked with arrowheads, and the r4/5 boundary arrowheads are labeled with a dot. ot, otic vesicle. (E-H) In situ hybridization of isl1 in blue and, in red, krox-20 in r3 and r5 and pax2a at the midbrain-hindbrain boundary (mhb). Arrowheads in F label early isl1-expressing nVII neurons in r4. isl1 also labels peripheral cranial ganglia, such as the trigeminal ganglion (tg in E). Anterior is towards the left. Somite stages are indicated (lower right-hand corners). Scale bars: in A, 50 µm in A-D; in E, 50 µm in E-H.

View larger version (129K): [in a new window]   Fig. 2. Zebrafish fgf3 expression overlaps with fgf8 expression in presumptive r4. In situ hybridization of fgf3 (in blue in A-E,H) and fgf8 (in blue in F,G and pink in H). Other markers are shown in red: pax2a at the MHB (A-G); no tail in the notochord (A); and krox-20 in r3 and r5 (B-G). Anterior is towards the top (A,B,F,H) or towards the left (C-E,G). In H, only the strong fgf8 expression in r4 is detectable in the double label (compare with F). Stages are indicated (lower right-hand corners). Scale bars: 100 µm in A,C; 50 µm in B,D-H. ntc, notochord; mhb, midbrain-hindbrain boundary; r3, rhombomere 3; r5, rhombomere 5; fb, forebrain; tb, tail bud.

View larger version (117K): [in a new window]   Fig. 3. FGF3 and FGF8 are required for r5 and r6 development. (A) In situ hybridization of various markers (in blue, named on the left) in wild-type (a,e,i,m,q), fgf3-MO (b,f,j,n,r), fgf8- (c,g,k,o,s) and fgf3-MO; fgf8- (d,h,l,p,t) embryos at 18 somites (a-h) and 20 somites (i-t). Markers in red are pax2a at the MHB (a-t) and krox-20 in r3 and r5 (e-t). In Figs 3, 4 and 8, wild-type and fgf3-MO embryos are siblings of fgf8- and fgf3-MO; fgf8- embryos, respectively. fgf8- and fgf3-MO; fgf8- embryos show loss of pax2a at the MHB. Dorsal views show anterior towards the left. n10 for each marker for each ‘genotype’. Scale bar: 50 µm. hb, midbrain-hindbrain boundary; r3, rhombomere 3; r5, rhombomere 5. (B) In situ hybridization of various markers (in blue, named on the left) in wild-type (a,e,i,m,q), fgf3-MO (b,f,j,n,r), fgf8- (c,g,k,o,s) and fgf3-MO; fgf8- (d,h,l,p,t) embryos at bud stage (a-d), five somites (e-l) and three somites (m-t). Markers in red are pax2a at the MHB and krox-20 in r3 and r5. pax2a at the MHB is still expressed, yet weakly and more broadly, in fgf8- and fgf3-MO; fgf8- embryos at these stages. Dorsal views show anterior towards the left. n7 for each marker for each ‘genotype’. Scale bars: in a, 50 µm in a-l; in M, 50 µm in m-t. mhb, midbrain-hindbrain boundary; r1-r7, rhombomeres 1-7; fb, forebrain.

View larger version (99K): [in a new window]   Fig. 4. FGF3 and FGF8 are required for neuronal development in r5 and r6. Confocal images of 48 h hindbrains stained with RMO-44, labeling reticulospinal neurons (A-D), anti-Islet, labeling motoneurons (E-H) or zn-8, labeling nVI and nIX motoneurons (I-L). Ventral views show anterior towards the top (A-H) or towards the left (I-L). Arrows in A,D indicate T interneurons. The large Mauthner neurons (M in A) are in r4. Arrows in E-H point to the rostral extent of nX neurons. n10 for each marker for each ‘genotype’. Scale bar: 50 µm. r2-r7, rhombomeres 2-7; V, nV neurons; VI, nVI neurons; VII, nVII neurons; IX, nIX neurons; X, nX neurons.

View larger version (88K): [in a new window]   Fig. 5. R4 transplants can induce expression of val and krox-20. (A) Diagrams of the transplantations. Putative r4 cells (positioned midway between the first somite boundary and the tip of the notochord) are excised from fluorescein-labeled donor embryos at early 1-somite stage and transplanted into either the r4 region of early 1-somite-stage hosts (left arrow) or ventral ectoderm of shield-stage hosts (right arrow). (B-B'') Host embryo (16 s) showing donor cells (B), krox-20 expression (B'), and merge of the two labels (B''). Most donor cells are in r4, some are in r5. (C) Host embryo (10 s) with r4 transplant in lateral ectoderm (arrow) stained for val (blue), krox-20 (red). (D-E'') Magnified view of transplant in C. (D) val (blue) and krox-20 (red) expression largely overlap (the krox-20-expressing cells express val, but the blue val is partially obscured). Donor cells (E) and krox-20 expression (E’) partially overlap (arrowhead) in a merge (E''), showing induction of krox-20 (and val, compare with D) in the host (arrow). 12/12 transplants showed some val and krox-20 expression in donor cells; three out of 12 showed some val and krox-20 expression in host cells. (F-G'') R4 transplant (in the ventral ectoderm of a 10 s host embryo) showing hoxb1a (blue) and krox-20 (red) (F), donor cells (G), krox-20 (G') and merge (G''). hoxb1a expression is found in donor cells (arrows) and in host cells. krox-20 expression is found in donor cells and host cells (arrowheads). Six out of eight transplants showed some hoxb1a and krox-20 expression in donor cells; two of these cases also showed hoxb1a and krox-20 expression in host cells; two out of transplants showed no hoxb1a or krox-20 expression associated with the transplant. (H-H'') R6 transplant (in the ventral ectoderm of a 10 s host embryo) showing donor cells (H) and val (H'). An overlay shows val expression within the transplant (H''). Anterior is towards the left. Scale bars: in B, 50 µm for B-B''; in C, 100 µm for C; in D, 50 µm for D-H''.

View larger version (116K): [in a new window]   Fig. 6. Wild-type, or val-, cells can partially rescue r5 krox-20 expression in the fgf3-MO; fgf8- hindbrain. (A) The transplantation technique used for the mosaic analyses. Naïve animal pole cells are excised from fluorescein-labeled donor embryos at shield stage and transplanted into the presumptive hindbrain region (Woo and Fraser, 1997) of shield stage fgf3-MO; fgf8- embryos. In some cases, fgf3-MO; fgf8-MO embryos were used as hosts. 100% (n=45) of the fgf3-MO; fgf8- or fgf3-MO; fgf8-MO control embryos (not receiving donor cells) showed no r5 krox-20 expression. (B) fgf3-MO; fgf8- control embryo (18 s) showing only r3 krox-20 expression. (C-C'') A host fgf3-MO; fgf8- embryo (18 s) showing wild-type donor cells (C), krox-20 expression (C') and merge of the two labels (C''). Red r5 cells (arrow, C'') are rescued fgf3-MO; fgf8- host cells expressing krox-20. Yellow r5 cells (arrowhead, C'') are wild-type donor cells expressing krox-20. (D-D') A host fgf3-MO; fgf8- embryo (18 s) showing krox-20 expression (D) and a merge (D') showing wild-type donor cells (green) and krox-20 expression (red). Donor cells populate the caudal hindbrain and anterior spinal cord and no rescue of r5 krox-20 is observed. (E-E') A host fgf3-MO; fgf8-MO embryo (2 s) showing krox-20 expression (E) and an overlay (E') of wild-type donor cells (green) and krox-20 expression (black) (E'). Rescue of r5 ege2 expression (arrowhead) has occurred adjacent to unilateral donor cells in r4 (arrow). Four out of nine embryos showed similar rescue at about 2 s. (F-F'') A host fgf3-MO; fgf8-MO embryo (18 s) showing val- donor cells (F), krox-20 expression (F') and merge of the two labels (F''). val- cells are excluded from the rescued r5 region on the right side of the hindbrain (arrow). val- cells are not excluded from the ‘r5’ level on the left, non-rescued side (arrowhead). Dorsal views show anterior towards the left. Scale bars: in B, 50 µm for B-D',F-F''; in E, 50 µm for E-E'.

View larger version (82K): [in a new window]   Fig. 7. fgf8 or fgf3 can induce expression of val and krox-20. (A) Embryo (14 s) with FGF8 bead (arrow). val (blue) and krox-20 (red) are induced in ectoderm adjacent to the bead, shown magnified in B. (C) Embryo (12 s) with FGF8 bead next to the caudal hindbrain, showing ectopic val (blue) and krox-20 (red). (D) Embryo (6 s) with fgf8-expressing cells (red, arrowhead) in r5. Ectopic krox-20 expression (blue, arrow) is induced in r6-r7. Seven out of 43 heat-shocked embryos showed ectopic krox-20 expression in the caudal hindbrain with nearby fgf8-expressing cells. (E) Embryo (6 s) with fgf8-expressing cells (red, arrowheads) in r6 and in ectoderm overlying r6-r7. Ectopic val (blue, arrow) spreads caudally into r7. Twelve out of 34 heat-shocked embryos showed ectopic val expression in the caudal hindbrain with nearby fgf8-expressing cells. (F) Embryo (6 s) with fgf8-expressing cells (red, arrowheads) in the midbrain and rostral hindbrain. val expression is not affected (blue). (G-G'') Embryo (8 s) with FGF3-Myc-expressing cell (arrowhead in G) in ectoderm overlying r5. Ectopic val spreads caudally into r7 (arrow in G') and ectopic krox-20 spreads caudally into r6 (arrow in G''). Ten out of 36 heat-shocked embryos showed ectopic val and krox-20 expression in the caudal hindbrain with nearby FGF3-expressing cells. Dorsal views show anterior towards the left. Scale bars: in A, 100 µm for A; in B, 50 µm for B,G-G''; in C, 50 µm for C; in D, 50 µm for D-F.

View larger version (10K): [in a new window]   Fig. 8. Incorporation of a new r4 signaling center in promoting hindbrain development. A zebrafish hindbrain showing seven rhombomeres (r1-r7). Many FGFs are expressed at the midbrain-hindbrain boundary, and zebrafish fgf8 is required for cerebellar/r1 development (reviewed by Rhinn and Brand, 2001). Retinoic acid likely signals from more posterior tissues (neural and/or mesodermal) (reviewed by Gavalas and Krumlauf, 2000). fgf3 and fgf8 expression overlap in r4 and promote the development of r5 and r6. FGF3 and FGF8 are required for otic development, but it is not clear how directly the FGF/r4 signals act on the head periphery. FGF3 and FGF8 from r4 may also have an influence on the development of more rostral rhombomeres (such as r3).