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First published online May 28, 2004
doi: 10.1242/10.1242/dev.01166

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Histone deacetylase 1 is required to repress Notch target gene expression during zebrafish neurogenesis and to maintain the production of motoneurones in response to hedgehog signalling

Centre for Developmental Genetics, School of Medicine and Biomedical Science, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK

View larger version (116K): [in a new window]   Fig. 1. Expression of hdac1 is widespread in the embryo at all stages of development and most abundant in the anterior CNS. Whole-mount in situ hybridisation of an hdac1 cDNA probe to zebrafish embryos at the (A) two-cell, (B) 1000-cell, (C) 80% epiboly, (D) 6-somite, (E) 18-somite and (F) 24 hpf stages. (G,J) Dorsal views of the hindbrain at 24 (G) and 48 (J) hpf. (H,K) Transverse sections through the hindbrain at the level of r3/r4, at 24 (H) and 48 (K) hpf. (I,L) Transverse sections through the anterior spinal cord at 24 (I) and 48 (L) hpf. Strongest expression of hdac1 is observed in the brain and spinal cord; by contrast, notochord and somites express much lower levels of hdac1 transcripts.

View larger version (108K): [in a new window]   Fig. 2. Mutation of hdac1 causes multiple developmental defects in the zebrafish embryo. (A,B) Bright-field images of live, 4-day-old hdac1hi1618 (A) sibling and (B) mutant embryos. Reduced brain size, absent jaw, curled down tail and pericardial oedema are clearly visible in the mutant. (C,D) Longitudinal sections of 3-day-old hdac1hi1618 (C) sibling and (D) mutant embryos taken at equivalent dorsoventral levels. Anterior is towards the left. Note the reduced size of the midbrain and anterior hindbrain and the dorsally open spinal cord in the mutant embryo. Position of midbrain-hindbrain boundary is marked with a black arrow. The hindbrain is indicated with a blue arrow and the row of somites (staining bright red) on the left side of each embryo is bracketed. Otic vesicles (lying below the plane of section in the sibling embryo) are marked with red arrowheads in D. (E,F) Transverse sections of 3-day-old hdac1hi1618 (E) sibling and (F) mutant embryos taken at equivalent anteroposterior levels passing through the otic vesicles and the heart. Note the absence of a heart valve (arrowhead) and semi-circular canal projections (arrow), and abnormal hindbrain shape in the mutant. (G,H) Transverse sections of 3-day-old hdac1hi1618 (G) sibling and (H) mutant embryos taken at equivalent anteroposterior levels passing through the pectoral fins. Note the absence of pectoral fin bud outgrowth (arrowhead) in the mutant.

View larger version (167K): [in a new window]   Fig. 3. Analysis of cell proliferation in the hindbrain of hdac1hi1618 (A,C,E) sibling and (B,D,F) mutant embryos between 25 and 38 hpf. Dorsal views of hindbrains from embryos at (A,B) 25, (C,D) 33 and (E,F) 38 hpf immunostained for the mitosis marker phospho-H3. Anterior is towards the left in all panels. At 25 hpf there are substantially fewer mitotic cells in the hindbrain of hdac1hi1618 mutants than in the hindbrain of sibling embryos. By 33 hpf, the numbers of mitotic cells in the hindbrain of mutant and sibling embryos are similar and they remain so at 38 hpf. For quantitation of hindbrain mitotic indices, see Table 1.

View larger version (115K): [in a new window]   Fig. 4. Mutation of hdac1 does not perturb primary neural patterning but neurogenesis is severely impaired. In situ hybridisation for expression of CNS patterning markers and proneural genes in hdac1hi1618 (A,C,E,G) sibling and (B,D,F,H) mutant embryos. (A,B) pax2a at 24 hpf marks the optic chiasm, midbrain-hindbrain boundary, otic vesicle and scattered spinal interneurones; (C,D) epha4 at 32 hpf marks the forebrain and hindbrain rhombomeres 1, 3 and 5; (E,F) ash1b and (G,H) ngn1 at 25 hpf mark neuronal precursors in brain and spinal cord. Expression of pax2a and epha4 in the embryonic CNS is unperturbed by the hdac1hi1618 mutation, whereas expression of the proneural genes ash1b and ngn1 is substantially reduced in hdac1hi1618 mutant embryos.

View larger version (114K): [in a new window]   Fig. 5. In situ hybridisation analysis comparing the expression domains of her6, ash1b and ngn1 in the hindbrain of hdac1hi1618 sibling and mutant embryos. her6 is aberrantly expressed in the medial hindbrain of hdac1hi1618 mutants at 26 and 33 hpf, and most strongly in rhombomeres 5 and 6 (arrows). By contrast, expression of ash1b and ngn1 in the hdac1hi1618 mutant hindbrain is almost completely extinguished. (A-F) her6; (G-L) ash1b; (M-R) ngn1 expression patterns. (A,B,G,H,M,N) Dorsal views of hindbrain, 26 hpf; anterior is towards the left. (C,D,I,J,O,P) Dorsal views of hindbrain, 33 hpf; anterior is towards the left. (E,F,K,L,Q,R) Transverse sections through rhombomere 5 of hindbrain, 33 hpf; dorsal is uppermost.

View larger version (94K): [in a new window]   Fig. 6. In situ hybridisation analysis comparing the expression domains of her6, ash1b and ngn1 in the dorsal diencephalon of hdac1hi1618 sibling and mutant embryos at 26 and 33 hpf. Loss of hdac1 function causes a stable expansion of the her6 expression domain in the dorsal diencephalon (arrows), as well as reductions in the diencephalic expression domains of ash1b and ngn1. (A-D) her6; (E-H) ash1b; (I-L) ngn1 expression patterns. In all panels, views are dorsal and anterior is towards the left.

View larger version (116K): [in a new window]   Fig. 7. Defective production and patterning of post-mitotic neurones and glia in the hindbrain of hdac1hi1618 mutant embryos. Immunohistochemistry for Hu-expressing post-mitotic neurones (A-F) and GFAP-positive glia (G,H) in the hindbrain of hdac1hi1618 sibling and mutant embryos at (A,B) 26, (C,D) 34 and (E-H) 38 hpf. Although the efficiency of neurone formation is reduced there is a progressive increase in the number of Hu-positive neurones in the hindbrain of hdac1hi1618 mutants between 26 and 38 hpf. However, these neurones are arranged in continuous longitudinal tracts extending through the hindbrain and a segmented arrangement is not adopted. Glia also fail to adopt their characteristic arrangement in each rhombomere and instead accumulate aberrantly in the anterior hindbrain.

View larger version (93K): [in a new window]   Fig. 8. Loss of hdac1 function disrupts specification and patterning of Isl1-expressing neurones. (A,B) Immunohistochemistry for Isl1-expressing neurones in the epiphysis of hdac1hi1618 (A) sibling and (B) mutant embryos. Note the absence of Isl1-positive cells in the anterior half of the mutant epiphysis (left of arrowhead). (C,D) Immunohistochemistry for Isl1-expressing branchiomotor neurones in the hindbrain of hdac1hi1618 (C) sibling and (D) mutant embryos. Note that the characteristic curved arrangements of nVII branchiomotor neurones spanning rhombomeres 4, 5 and 6 in the sibling hindbrain are absent in the mutant hindbrain and replaced by two small clusters of Isl1-positive neurones in rhombomere 4 (arrows). Trigeminal (nV) motoneurones in rhombomere 2 are marked (arrowheads). (E-H) Expression of an Isl1-GFP transgene reveals the position and morphology of differentiated branchiomotor neurones in embryos microinjected with (E,F) control MO or (G,H) hdac1-MO, at (E,G) 30 hpf and (F,H) 36 hpf. Positions of otic vesicles are marked with a white oval and red asterisk. In A-H, views are dorsal, anterior is towards the left. At both 30 and 36 hpf, hdac1-MO-injected embryos exhibited the same four, small clusters of differentiated branchiomotor (trigeminal and facial) neurones with axons that projected anteriorly, and there were no Isl-GFP-positive neurones located posterior to rhombomere 4. By stark contrast, control MO-injected embryos developed a normal population of Isl1-GFP-expressing branchiomotor neurones which increased in size and morphological complexity between 30 and 36 hpf (I,J) Immunohistochemistry for Isl1-expressing neurones in the spinal cord of hdac1hi1618 (I) sibling and (J) mutant embryos, and wild-type WIK embryos microinjected with (K) a control MO or (L) an hdac1-MO, at 24 hpf. Lateral views, anterior is towards the left. Isl1-positive motoneurones lie in the ventral spinal cord; Rohon-Beard cells are located in the dorsal spinal cord and stain relatively strongly for Isl1 protein. Homozygosity for the hdac1hi1618 mutation or microinjection of an hdac1-MO reduces the number of Isl1-expressing neurones formed in the spinal cord.

View larger version (149K): [in a new window]   Fig. 9. The hdac1 morphant CNS phenotype is epistatic to that of the mind bomb mutant. (A,E,I,M,Q) Unaffected mib siblings injected with control MO. (B,F,J,N,R) mib homozygous mutant embryos injected with control MO. (C,G,K,O,S) mib siblings injected with hdac1-MO. (D,H,L,P,T) mib homozygous mutant embryos injected with hdac1-MO. (A-H) In situ hybridisation analysis of her6 expression in (A-D) dorsal diencephalon (30 hpf) or (E-H) hindbrain (30 hpf). Loss of mib function reduces the abundance of her6 transcripts in the dorsal diencephalon and hindbrain, whereas loss of hdac1 function derepresses her6 in the dorsal diencephalon and hindbrain both in mib-siblings and mutants. (I-L) In situ hybridisation analysis of ngn1 expression in 25 hpf embryos. Loss of mib function increases ngn1 expression throughout the CNS, whereas loss of hdac1 function abolishes ngn1 expression in the CNS both in mib-siblings and mutants. (M-T) Immunohistochemistry for Isl1-expressing (M-P) epiphysial and (Q-T) branchiomotor neurones in the hindbrain of 30 hpf embryos; dorsal views, anterior is leftwards. Loss of mib function produces supernumerary Isl1-positive cells in the epiphysis and throughout the hindbrain, whereas loss of hdac1 function severely impairs the production of Isl1-positive cells both in mib-siblings and mutants.

View larger version (79K): [in a new window]   Fig. 10. The hedgehog signalling pathway is intact in hdac1 mutant embryos but motoneurone precursors require wild-type levels of hdac1 activity to respond to elevated levels of hedgehog signalling. (A-D) In situ hybridisation analysis of (A,B) shh and (C,D) ptc1 expression in hdac1hi1618 (A,C) sibling and (B,D) homozygous mutant embryos at 24 hpf. Positions of zli are indicated (arrowheads). Loss of hdac1 function does not significantly affect shh expression in ventral midline cells of hindbrain and spinal cord, and expression of the shh target gene ptc1 is relatively unpertubed in CNS and paraxial mesoderm. (E-L) Expression of an Isl1-GFP transgene reveals the position and morphology of branchiomotor neurones in 30 hpf embryos microinjected with (E,G) control MO; (F,H) hdac1-MO; (I,K) control MO plus 100 pg shh mRNA; (J,L) hdac1-MO plus 100 pg shh mRNA. (E,F,I,J) Dorsal views of hindbrain, anterior is towards the left; (G,H,K,L) lateral views of hindbrain, anterior is towards the left. Overexpression of shh dramatically increased the number of Isl1-GFP-positive branchiomotor neurones. No supernumerary branchiomotor neurones were formed when an hdac1-MO was co-injected with shh mRNA and only the rudimentary pattern of Isl1-expressing neurones characteristic of hdac1-deficient embryos was observed.

View larger version (128K): [in a new window]   Fig. 11. Sustained production of smoothened-dependent branchiomotor neurones requires wild-type levels of hdac1 activity. Immunohistochemistry for Isl1-expressing branchiomotor neurones in the hindbrain of smohi1640 (A,B,E,F) sibling and (C,D,G,H) homozygous mutant embryos, at (A-D) 26 hpf and (E-H) 32 hpf. Embryos were microinjected with either (A,C,E,G) control MO or (B,D,F,H) hdac1-MO. Dorsal views, anterior is towards the left. Specification of all Isl1-expressing branchiomotor neurones requires smoothened function. Initial specification of Isl1-positive trigeminal motoneurones at 26 hpf was hdac1 independent, but further production of smoothened-dependent branchiomotor neurones was impaired in hdac1-deficient embryos and only two further small clusters of Isl1-positive facial neurones had formed by 33 hpf, in contrast to the situation in control-MO-injected embryos.

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