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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00662

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Tracing of her5 progeny in zebrafish transgenics reveals the dynamics of midbrain-hindbrain neurogenesis and maintenance

Zebrafish Neurogenetics Junior Research Group, Institute of Virology, Technical University-Munich, Trogerstrasse 4b, D-81675 Munich, Germany GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany

View larger version (25K): [in a new window]   Fig. 1. Schematic organization of the MH domain at the 10-somite stage (A,C) and at 24 hpf (B). All views are anterior towards the left; A and C are dorsal and ventral views of the alar and basal plates, respectively; B is a sagittal view, the broken line delimiting the alar/basal boundary. The early MH domain comprises the mes- and metencephalic vesicles; the contribution of each vesicle to the late MH derivatives, as demonstrated in transplantation experiments in the avian embryo (Hallonet and Le Douarin, 1990; Hallonet et al., 1993; Martinez and Alvarado-Mallart, 1989) (and without considering the floor and roof plates) is color-coded and indicated by the vertical lines: (1) the alar plate of the mesencephalic vesicle contributes to the tectum; (2) in addition, the caudal third of the mesencephalic vesicle is at the origin of the alar part of the isthmus and dorsomedial part of the cerebellar plate (future vermis) and alar part of r2; (3) the alar plate of the metencephalon gives rise to the lateral cerebellum (future hemispheres); (4) the basal plate of the mesencephalic vesicle gives rise to the tegmentum; (5) the basal plate of the metencephalic vesicle gives rise to the pons (basal r1) and basal plate of r2. The isthmus is colored in yellow. Its basal part has not been precisely mapped and was not studied for its inductive properties of MH fate; it is drawn here based on the expression pattern of isthmic organizer markers such as wnt1 and fgf8. The `intervening zone' is defined as the territory delayed in neurogenesis (Geling et al., 2003). It is located at the MHB but its spatial relationship with the isthmus has not been established. Cb, cerebellum; Di, diencephalon; Is, isthmus; IZ, intervening zone; Mes, mesencephalon; Met, metencephalon; Myel, myelencephalon; Po, pons; r, rhombomere; Tc, tectum opticum; Tg, tegmentum.

View larger version (51K): [in a new window]   Fig. 2. Structure of the her5 genomic locus and reporter constructs and corresponding GFP expression. (A) Construction of her5PAC:egfp by ET-cloning-mediated recombination of the egfp cDNA within exon 2 of her5. The her5 locus comprises 3 exons (blue), of which exon 2 encodes the basic and first helix domain of the Her5 protein (bHLH domain labeled in red as b, H1, L and H2). Recombination arms (a',b') matching exon 2 were amplified in frame with the egfp sequence and a floxed zeocine resistance cassette (zeo) (top construct). The resulting product was inserted in vitro within a her5-containing PAC by ET-mediated homologous recombination (Muyrers et al., 2000; Muyrers et al., 1999). The zeo cassette was subsequently deleted by Cre excision in vitro, generating the herPAC:egfp construct (bottom line). (B) Reporter constructs used to localise her5 regulatory elements in transient (black lines) or transgenic (red lines) assays. Most constructs were generated from her5PAC:egfp (bottom construct) by PCR amplification and contain egfp in frame within her5 exon 2. Numbering to the left of each fragment refers to the length of upstream sequence from the transcriptional start site, in bp. The expression profile driven by each construct is written to the right. Note that the enhancer element(s) driving endodermal expression are located within 240 bp of upstream sequence and/or intron 1, and that sequences driving specific MH expression are recovered with 2.9 kb of upstream sequence. (C) Endogenous her5 transcription at 70% epiboly (onset of neural her5 expression) revealed by whole-mount in situ hybridisation (blue staining). her5 is expressed in a V-shaped domain at the AP level of the MH anlage (MH) and in a subset of anterior endodermal precursors (e) (see also Bally-Cuif et al., 2000). (D-H) Selected examples of GFP protein expression driven by representative reporter constructs [bright field (top) and fluorescent (bottom) views of transgenic embryos, constructs as indicated below each panel]. All constructs illustrated drive expression to the anterior endoderm. Constructs comprising more than 2.9 kb of upstream sequence (D,E) drive selective neural expression to the MH. Intermediate constructs (F,G) drive unrestricted anterior neural expression.

View larger version (85K): [in a new window]   Fig. 7. MH precursors are maintained in ace and noi mutants. (A-D) Double in situ hybridisation for egfp (red) and her5 (blue) in her5PAC:egfp transgenic wild-type, ace and noi siblings at the stages indicated demonstrates that egfp transcription also reproduces her5 expression in ace and noi, and is downregulated following a correct schedule during the MH maintenance phase. (E-G) Live observation of her5PAC:egfp transgenic wild-type, ace and noi siblings under fluorescence microscopy at 24 hpf reveals that most descendants of early her5-positive cells (positive for GFP protein, green) are maintained, although MHB identities, such as cranial motoneurons III and IV (revealed using the isl1:gfp transgene, insets) (Higashijima et al., 2000) are missing. (H-M) Analyses of apoptosis (H-J, Acridine Orange staining) and cell division (K-M, anti-phosphohistone H3 immunocytochemistry, brown staining) demonstrate that the pattern of cell death and proliferation are comparable in the MH area (bar) in wild-type, ace and noi siblings at least until the 15-somite stage. Embryos in K-M are double stained for wnt1 expression, which is strongly downregulated in ace and absent in noi at that stage (blue staining, arrowheads).

View larger version (32K): [in a new window]   Fig. 5. The earliest her5-positive domain at gastrulation contains an ordered distribution of MH precursors and prefigures the later MH domain. (A) Experimental approach. The earliest her5-positive domain (schematized in blue on a dorsal view of the neural plate at 70% epiboly, left panel) is reflected by GFP protein expression starting at 95% epiboly (green, right panel). Thus, the anterior and posterior extremities of the early her5-positive domain were fate mapped by laser activation of caged fluorescein within the most anterior or posterior GFP-positive cell rows at 95% epiboly (yellow and red dots, respectively). (B-E) Location of cells activated in A, revealed at 24 hpf by whole-mount anti-fluorescein immunocytochemistry (brown staining) (all embryos anterior leftwards, with black arrow to the midbrain-hindbrain boundary). (B,C) Anterior activations give rise to cell clones distributing within the anterior midbrain (two different embryos are shown, brackets to the midbrain, yellow arrows to delimit the cluster of uncaged cells). (D,E) Posterior activations produce cell clones located posterior to the midbrain-hindbrain boundary and populate r2 (two different embryos are shown, brackets to r1 and r2, red arrows to delimit the cluster of uncaged cells). mid, midbrain; r1-2, rhombomeres 1-2.

View larger version (73K): [in a new window]   Fig. 3. Comparison of endogenous her5 (blue) and gfp (red) RNA transcription profiles in her5PAC:egfp (A,C,D) and -3.4her5:egfp (B,E,F) transgenic embryos, at the stages indicated. All views are high magnifications of the MH area in flat-mounted embryos, dorsal (A,B,E,F and inset in D) or sagittal (C,D) orientations, anterior towards the top (A,B) or left (C-F). Endogenous her5 and gfp expressions exactly coincide at all embryonic stages, including the initiation (A,B) and maintenance (C-F) phases of her5 transcription, demonstrating that all the regulatory elements driving MH her5 expression are contained within the her5PAC:egfp and -3.4her5:egfp constructs.

View larger version (77K): [in a new window]   Fig. 4. The distribution of GFP protein in her5PAC:egfp embryos reveals the fate of endodermal and neuroectodermal cells expressing her5 at gastrulation. GFP protein in her5PAC:egfp embryos was observed on live specimen (J) or revealed by immunocytochemistry (A-I, brown DAB staining; and K-Q, green FITC staining) at the stages indicated (bottom left of each panel). (H-J) Whole-mount views: (H,J) dorsal views, anterior leftwards; (I) lateral view, anterior leftwards. (K-Q) Sagittal sections, anterior leftwards. In K,L,O-Q, the top and bottom panels are bright-field and fluorescent views, respectively, of the same sections that were each processed for in situ hybridisation (top panels, blue staining, probes indicated in the bottom right-hand corner) and immunocytochemistry against GFP protein (bottom panels). (M,N) High magnifications of levels equivalent to those boxed in K and L, respectively (red arrows indicate rhombomere boundaries). Overlay pictures of the in situ hybridisation staining (revealed using Fast Red, red fluorescence) and GFP immunocytochemistry (FITC staining). The cytoplasm of cells doubly positive for GFP protein and for the in situ hybridisation marker (hoxa2 or krox20, respectively) appears yellow. The descendants of endodermal her5-expressing cells distribute to the entire AP and mediolateral extent of the pharynx (A; cross-section at hindbrain level in B, bottom). At 12 somites, the descendants of neural her5-expressing cells distribute over a broad domain at the level of the MH (A; cross-section at forebrain level in B, top). Neural crest cells that exit the MH are also GFP-positive (C, note a dorsal stream and a stream caudal to the eyes, and cross-section at forebrain level in D). In E-J, arrows indicate the midbrain-hindbrain boundary; note that GFP protein distributes posterior to this level (i.e. to metencephalic derivatives) until 24 hpf, and encompasses r2 (K,O,Q; blue and green arrowheads to the anterior limit of hoxa2 expression; green arrows to GFP-positive cells in r2), with a minor contribution to r3 and r4 before the 20-somite stage (L,P; white brackets indicate r3 and r5, green arrow in L indicates GFP cells in r4; green arrowhead in P indicates the posterior limit of GFP extension at the r2/r3 boundary). At 10 somites, GFP-positive cells in r2 and r3 co-express hoxa2 and krox20, respectively (yellow arrows in M,N). e, endoderm; hg, hatching gland; MH, midbrain-hindbrain domain; MHB, midbrain-hindbrain boundary; nc, neural crests streams.

View larger version (74K): [in a new window]   Fig. 8. MH precursors display altered molecular identities in ace and noi mutants. (A-H') Comparison of GFP protein (anti-GFP immunocytochemistry, brown staining) and pax6.1 RNA (ISH, blue staining) at the stages indicated in sagittal sections of her5PAC:egfp transgenic wild-type (A,C,E,G), ace (B,B',F,F',H,H') and noi (D) embryos. B', F' and H' are magnifications of the areas boxed in B, F and H. Note that GFP protein and pax6.1 expression are never co-expressed anteriorly in wild type (A,C,E,G) and noi (D), while extensive overlap between the two stainings is present in ace at the 15-, 20- and 30-somite stages (F',H',B'). (I-K,M-O) Comparison of GFP protein (anti-GFP immunocytochemistry, bottom panels, green staining) and fgfr3 (I-K) or otx2 (M-O) RNAs (in situ hybridisation, top panels, blue staining) at the stages indicated in her5PAC:egfp transgenic wild-type (I,M), ace (J,N) and noi (K,O) embryos. Top and bottom panels are bright-field and fluorescence views, respectively, of the same sagittal sections. Green arrowheads on the bright-field pictures point to the limits of GFP protein distribution. Note in ace that anterior GFP-positive cells do not co-express fgfr3 (J, compare with I), and that posterior MH cells are all otx2-positive (N, compare with M). By contrast, in noi, all the descendants of MH precursors express fgfr3 (K) but an otx2-negative territory is maintained within the caudal GFP-positive population (O). (L) Expression of fgfr3 revealed by whole-mount in situ hybridisation shows that MH precursors in noi are fgfr3-positive already at the 15-somite stage (bar, bottom panel, compare with wild-type sibling, top panel).

View larger version (74K): [in a new window]   Fig. 6. Dynamic regulation of her5 expression within the MH domain. (A-O) Comparison of her5 expression (revealed by in situ hybridisation, blue staining in C-E,G,I,K, red staining in M,O) and GFP protein distribution (direct visualization under fluorescence microscopy, green in A,B; or revealed by anti-GFP immunocytochemistry, brown staining in D or green staining in F,H,J,L,N,O) in her5PAC:egfp embryos at the stages indicated. (A-D) Whole-mount views (A, dorsal, anterior leftwards; B,C, lateral, anterior leftwards; D, dorsal view of a hemi-neural plate, anterior upwards); E-O are sagittal sections, all views focus on the MH domain and are oriented anterior towards the left. The MHB is indicated by a red arrow at all stages where it is morphologically visible. (E-L) Bright field (top panels) and fluorescent (bottom panels) views of the same sections; M-O are red, green or double fluorescent views of the same section. Note the dramatic difference in the extent of her5 transcripts (C) and GFP protein (A,B) along the AP axis at 24 hpf. Because egfp transcription faithfully reproduces her5 expression in her5PAC:egfp embryos (Fig. 3), whereas GFP protein is stable, this demonstrates that her5 expression is lost from progeny cells over time. This process is progressive (D-L) and sequential: it involves first a restriction of her5 expression in the posterior aspect of the MH domain (blue and brown arrows indicate the limits of her5 RNA and GFP protein staining, respectively, in D; blue dots indicate the posterior limit of her5 transcription. Note that the two limits coincide anteriorly but differ by one or two cell rows posteriorly). At three somites, her5 restriction begins in ventral and lateral aspects of the mesencephalon (black arrows in E,G), and continues after 16 somites (I) along the dorsal midline (blue arrows in E,G indicate maintained dorsal expression of her5 prior to that stage). Note that in M-O, the final her5 expression domain is located in the center of the GFP-positive territory, demonstrating that her5 expression gets restricted in a converging manner towards the MHB. (P) Resulting model for the regulation of her5 expression and the progression of neurogenesis between 70% epiboly (a), 90% epiboly (b) and 30 somites (c,d) in the MH domain [combined from the present data and data from Geling et al. (Geling et al., 2003)]. her5 expression at 70% epiboly (blue), traced using GFP protein in her5PAC:egfp embryos, is the entire MH anlage (green lines and labeling, 45-50 cell rows at 30 somites). Between 70 and 90% epiboly (b), her5 expression is lost from progeny cells posteriorly (compare green lines and blue). At 90% epiboly, her5 expression is adjacent to the first anterior neurogenesis sites: the ventrocaudal cluster (vcc, pink, precursor of the nucleus of the medial longitudinal fascicle, nMLF) and future motor and sensory neurons of r2 (orange) (see Geling et al., 2003). At 30 somites (c), her5 expression has been dramatically lost upon cell divisions and is restricted to three to five cell rows at the MHB. Correlatively (d), neurogenesis (revealed by zcoe2 expression) (Bally-Cuif et al., 1998), still adjacent and non-overlapping with her5 expression (compared c with d), progressed towards the MHB (red arrows) (embryo with the same orientation as in c, focus on the basal plate).

View larger version (53K): [in a new window]   Fig. 9. Schematic representation of the fate of MH precursor cells (green, territory delimited by the green stars) in wild-type embryos (A) or in the absence of Fgf8 (B) or Pax2.1 (C) activities (interpreted from Fig. 8, and data not shown). In each drawing, the thin horizontal black line delimits the alar/basal boundary; gene expressions are color coded. Pink arrows delimit the population of anterior MH cells that acquires a pax6.1-positive identity in ace and blue arrows point to the extension of fgfr3 expression in noi. Note the striking differences in the alternative identities taken by MH precursors depending on the mutant context.