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doi: 10.1242/10.1242/dev.00375

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bHLH transcription factor Her5 links patterning to regional inhibition of neurogenesis at the midbrain-hindbrain boundary

Andrea Geling1,*, Motoyuki Itoh2,*, Alexandra Tallafuß1, Prisca Chapouton1, Birgit Tannhäuser1, John Y. Kuwada3, Ajay B. Chitnis2,{dagger} and Laure Bally-Cuif1,{dagger}

1 Zebrafish Neurogenetics Junior Research Group, Institute of Virology, Technical University-Munich, Trogerstrasse 4b, D-81675 Munich, Germany and GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany
2 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
3 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA



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  		Fig. 2. Her5 as a candidate to control IZ formation. (A-C) Whole-mount in situ hybridization at the 3-somite stage (A, dorsal view, anterior to the top) and at 24 hpf (B,C, sagittal views, anterior to the left) with the markers indicated (bottom left, color-coded in A). Note that her5 expression in wild-type embryos delineates the IZ (bracket) from the onset of neurogenesis (A) until at least 24 hpf (IZ identified by the gap in zcoe2 staining in B). (D) Structures of the wild-type and mutant forms of her5 cDNA and their encoded proteins used for functional assays. Top: full-length her5 cDNA as determined from our genomic analyses (A.T. and L.B.-C., unpublished), which starts at ATG1 and encodes nine additional N-terminal amino acids compared to the published sequence (Müller et al., 1996Go) (see box for protein sequence). Bottom: hsp-her5 construct used to generate transgenic lines for conditional misexpression; this construct is built from the clone of Müller et al. (Müller et al., 1996Go) such that the first ATG is deleted and the second ATG is used for the generation of an otherwise fully functional Her5 protein (see Materials and Methods). As a control, a morpholino directed against ATG2 (MOtg, inset) inhibits translation of the transgene mRNA but not that of the endogenous her5 (data not shown, see Materials and Methods). For loss-of-function experiments, a morpholino directed against ATG1 (MOher5, inset) was used, which inhibits the function of the endogenous Her5 mRNA. Abbreviations as Fig. 1 plus, b, basic DNA-binding motif; HLH, helix-loop-helix dimerization motif; IZ, intervening zone; mes, mesencephalon; r1, rhombomere 1.

 


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  		Fig. 3. Her5 is necessary and sufficient to control IZ formation. (A-C) Reliability of the hsp-dependent expression system. (A,B) Embryos from a cross between parents heterozygous for the hsp-her5 transgene probed for her5 expression (in situ hybridization) before (A) and after (B) a 1- hour heat-shock. While no ectopic expression of her5 is detected without heat-shock, ectopic her5 expression is ubiquitously induced upon heat-shock (white arrows indicate endogenous her5 expression at the MHB, black arrows indicate hsp-driven ubiquitous expression). (C) Stability of the induced her5 mRNA upon heat-shock, determined by whole-mount in situ hybridization (in percentage of the estimated intensity of staining that immediately follows a 0.5-hour heat-shock pulse). Induced mRNAs become undetectable within 1.5 hours following the end of the heat-shock. (D-H,N,O) Ectopic expression of Her5 inhibits ngn1 expression in the vcc and presumptive r2MN. Whole-mount in situ hybridization or immunocytochemistry with the markers indicated (bottom left, color coded) on transgenic embryos (tg) (E,F,H,O) and their wild-type siblings (wt) (D,G,N) at the 3-somite (D-F), 20-somite (G,H), and 36 hpf (N,O) stages, following a 1-hour heat-shock at late gastrulation (hs). D-F and N,O are dorsal views of the MH area in flat-mounted embryos, anterior to the top; G,H are sagittal views of the head, anterior to left; the insets show unperturbed ngn1 expression in the spinal cord. The misexpression of her5 during late gastrulation inhibits ngn1 expression in the vcc and r2MN at the 3- somite stage (white asterisks in E). Non-heat-shocked transgenics display a ngn1 profile indistinguishable from non-transgenic controls (not shown). This effect is maintained until at least the 20-somite stage (H), and is rescued upon injection of MOtg, a morpholino oligonucleotide selective of the transgene (F). At 24 hpf, the number of nMLF neurons (brown arrows), which derive at least in part from the vcc, is also significantly reduced in hsp-her5 transgenics (O) (red arrow to her5 expression at the MH junction). (I) Genotyping results to identify transgenic embryos in H (PCR for the transgene). Lane 1: embryo H, lane 2: embryo G, lane 3: negative control, lane 4: positive control. An identical procedure was used to identify embryos in N,O. (J-M,P,Q) The inhibition of Her5 activity leads to the differentiation of ectopic neurons in place of the IZ. J,K: dorsal views of the MH area in flat-mounted embryos at the 3-somite stage, anterior to the top, probed for ngn1 expression following injection of MOher5, a morpholino selective of endogenous her5 (K), compared to non-injected wild-type control embryos (J). Note that the vcc and r2MN clusters are bridged (double arrow), while other neuronal populations (e.g.r4MN, arrowhead) are unaffected. (L,M) TUNEL assay in wild-type (L) and MOher5-injected (M) embryos shows that injections are not followed by increased apoptosis in the MH area (bar). (P,Q) At 36 hpf, an ectopic HNK1-positive neuronal cluster (brown arrows) lies across the MH junction (identified by her5 expression, blue arrow) upon MOher5 injection. Note reduced her5 levels at the MHB in Q (compared with P), a late event suggesting indirect positive autoregulation of her5 expression. IZ, intervening zone; MH, midbrain-hindbrain domain; nMLF, nucleus of the medial longitudinal fascicle; r2MN, motorneurons of rhombomere 2; r4MN, motorneurons of rhombomere 4; vcc, ventrocaudal cluster.

 


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  		Fig. 1. The intervening zone (IZ) displays intrinsic mediolateral differences and is shaped by antagonistic activities from neurogenesis-promoting signals and the IsO. Whole-mount in situ hybridization at the 3-somite stage (A,D-F,K,L) (dorsal views, anterior to the top) and 24 hours post-fertilization (hpf) (B,C,G-J) (sagittal views, anterior to the left) with the markers indicated (bottom left, color-coded). (A-C) Intervening zone (IZ) location in wild-type (wt) embryos. At the 3-somite stage, the IZ separates the ventro-caudal cluster (vcc) from the r2 motor (r2MN) and sensory neurons (r2S), and encompasses most of the MH primordium, as revealed by pax2.1 expression. By 24 hpf, the IZ (bracket in B,C) has narrowed to a stripe at the MHB. (D-F) Intrinsic differences between the neurogenic capacities of lateral versus medial domains of the IZ. Upon injection of 25 pg ngn1 mRNA at the 1-cell stage (ngn1-inj, E), ectopic neurogenesis is induced within the neural plate outside proneural clusters (arrows in E) including the basal domain of the IZ (bracket), while the IZ remains neurons-free in lateral regions. 125 pg ngn1 (F) are necessary to force neurogenesis within the IZ alar domain (arrow in F; location of the IZ in F is indicated by the bracket). This phenotype is correlated with the loss of expression of the MH markers pax2.1 and wnt1 at 24 hpf (G-J, red arrows). Note that the profile of pax2.1 and wnt1 expression is otherwise unaltered (pax2.1: optic chiasm, hindbrain interneurons: G,H, black arrows. wnt1: midbrain dorsal midline, rhombic lips: I,J, black arrows). (K,L) The anterior-to-posterior extent of the IZ correlates with IsO activity, and is enlarged in hdl mutants, which overactivate Wnt signaling (L compared with K, bracket). Scale bars: 0.1 mm. IZ, intervening zone; vcc, ventrocaudal cluster; r2MN, rhombomere 2 motorneurons; r4MN, rhombomere 4 motorneurons; r2S, rhombomere 2 sensory neurons; delA, delta A; ngn1, neurogenin 1.

 


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  		Fig. 4. Her5 activity can inhibit ngn1 until 24 hpf, in a dose-dependent manner, but does not affect HuC expression. Whole-mount in situ hybridization for ngn1 (blue) and her5 (red), with immunocytochemistry for HuC protein expression (brown), at the stages indicated (bottom left) following a two-hour heat-shock (hs) between the 8- and 15-somite stages (A-G), the 15- and 20-somite stages (H,I) and at 24 hpf (J,K) in hsp-her5 transgenic heterozygotes (+/tg), homozygotes (tg/tg) or their non-transgenic siblings (wt). Dorsal views of flat-mounted anterior neural tubes (A-E,H-K) or tails (F,G), anterior to the top; bracket indicates the IZ, vertical black bar indicates the MH domain. ngn1 expression is downregulated in a dose-dependent manner immediately after heat-shock at any of these stages (B,C,I). This phenotype is stable in the MH domain (blue arrows to restored ngn1 expression in the fore- and hindbrain in E). In contrast, neuronal precursors already expressing HuC are only moderately affected (brown arrows in F-I) [HuC is first detectable in vcc neurons at about the 20-somite stage (H,I), thus at the 15-somite stage we focused on HuC expression in the tail (F,G)[. At 24 hpf, only the MH domain is sensitive to ectopic Her5 activity (blue arrows to unaffected ngn1 expression in the fore- and hindbrain in K). IZ, intervening zone; hs, heat-shock; som, somites.

 


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  		Fig. 5. Her5 activity does not control MH regional patterning. Whole-mount in situ hybridization at the 3-somite (E-H), 5-somite (A-C), and 24 hpf (D) stages for the expression of patterning and neurogenesis markers, as indicated (bottom left, color coded), following up- or down-regulation of Her5 activity. All panels are dorsal views of whole-mount (E-G) or flat-mounted (A-D,H) embryos, anterior to the top; arrows point to the MH junction. (A-D) MH patterning is not altered in transgenic hsp-her5 embryos (right in each panel) compared to non-transgenic siblings (left) by heat-shock during late gastrulation (A-C) or by repetitive heat-shocks (D). (E-G) MH patterning also remains unaltered in MOher5-injected embryos (right in each panel) compared to controls (left). (H) Co-expression of ngn1 and pax2.1 (see also fluorescent view, inset) across the MHB in a single embryo upon MOher5 injection demonstrates that neurogenesis and patterning can be uncoupled by Her5 activity.

 


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  		Fig. 6. Her5 activates cell proliferation within the MH domain, but this process is in itself insufficient to account for the regulation of ngn1 expression. (A,F) her5 expression (in situ hybridization, blue staining) and density of cells in M phase (brown anti-phosphoH3 immunostaining) (in number of positive cells per cell row) in the anterior neural plate at the 3-somite stage (in territories schematized in A) in hsp-her5 transgenics (C), their wild-type siblings (B) (both heat-shocked), wild-type (D) and MOher5-injected embryos (E). A is a schematic representation of the neural plate in B,C; B-E are flat-mounted views of the anterior neural plate (B,C) or the endogenous her5 domain (D,E), anterior to the top. White arrow in B,C, the endogenous domain of her5 expression (territories A + B); box in D,E indicates territory A. Proliferation is enhanced in territory A compared to other neural plate domains in wild-type embryos (brown arrows in B,D). Her5 is sufficient to increase proliferation within the MH domain upon ectopic expression (territories A-D) (F, left panel), and is necessary for the increased level of proliferation of territory A (F, right panel). (G-J) Expression of the cyclin-dependent kinase inhibitor-encoding gene p27Xic1-a is downregulated by Her5 within the IZ. Expression of p27Xic1-a and pax2.1, as indicated (bottom left, color-coded), in hsp-her5 transgenic embryos after heat-shock (H) and MOher5-injected embryos (J) compared to their wild-type siblings (G,I) at the 3-somite stage. In the vcc and IZ, p27Xic1-a expression is strikingly similar to that of ngn1 (e.g. Fig. 3D). p27Xic1-a expression is down-regulated within the neural plate following ectopic her5 expression (arrow in H), and is activated across the IZ when Her5 activity is blocked (white arrows in J). Concomitantly in the latter case, p27Xic1-a expression is partially reduced in the vcc area, a phenomenon at present unexplained but independent of cell migration (A.G. and L.B.-C., unpublished data). (K-N) The direct inhibition of cell proliferation does not affect IZ formation and her5 expression. Expression of ngn1 or her5 (blue in situ hybridization staining) and anti-phosphoH3 immunostaining (brown nuclei) at the 3-somite stage in embryos treated with the cell proliferation inhibitor aphidicolin at the onset of neurogenesis (L,N) compared to mock-treated siblings (K,M). Although phosphoH3 staining is virtually abolished upon aphidicolin treatment, both IZ size (bracket in K,L) and her5 expression appear normal. Aphi, aphidicholin-treated embryo; hs, heat-shocked embryo; IZ, intervening zone; tg, transgenic; vcc, ventrocaudal cluster.

 




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