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doi: 10.1242/10.1242/dev.00452

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Ash1a and Neurogenin1 function downstream of Floating head to regulate epiphysial neurogenesis

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (94K): [in a new window]   Fig. 1. ash1a, ngn1 and neuroD are expressed in spatially and temporally different populations of cells in the epiphysis. Lateral views of whole brains with anterior to the right, showing expression of ash1a, ngn1 and neuroD at 8-, 12-, 18- and 24-somite stages. Stage is indicated on the left and probe above. Arrows indicate the location of the epiphysis and arrowheads indicate the anterior and posterior limits of the epiphysis, which is delineated by the dashed lines (J,K). ash1a was expressed both earlier and more broadly than ngn1 in the presumptive epiphysis, whereas neuroD was expressed later than both ash1a and ngn1. npc, nucleus of the posterior commissure; t, telencephalon; d, diencephalon. Scale bar: in A, 50 µm for A-I; in J, 10 µm for J,K.

View larger version (78K): [in a new window]   Fig. 2. flh regulates the expression of ash1a, ngn1 and neuroD in the epiphysis. Dorsal views of whole brains with anterior at the top, showing expression of ash1a, ngn1 and neuroD in the epiphysis of wild-type (WT) and flh-mutant embryos at 14- and 24-somite stages. Genotype is indicated above, genes analysed on the left of the panels, and stage in the bottom right of each image. Flh is required for the maintenance of ash1a expression (D) and for the activation of ngn1 and of neuroD expression (F,J). Scale bar: 15 µm.

View larger version (92K): [in a new window]   Fig. 3. ash1a and ngn1 are important regulators of neurogenesis in the epiphysis. A-F and I-L are dorsal views of the epiphysis with anterior at the top. G and H are lateral views of the brain with anterior to the right. (A-F) Expression of isl1 at the 25-somite stage in the epiphysis of wild-type (WT), ash1a MO-, ash1a5'UTR MO-, ngn1MO-, ash1a and ash1a5'UTRMO-, or ash1a and ngn1 MO-injected embryos. Neuronal production was reduced in ash1a morphants (B,C,E) but was normal in the ngn1 morphant (D). A stronger effect was observed in the double ash1a/ngn1 morphant (F) compared with single ash1a morphants (B,C,E). A combination of ash1a5'UTR MO and ash1a MO gave rise to a similar phenotype (E) to the single ash1a or ash1a5'UTR morphants (B,C). (G,H) Expression of isl1 at 25 hours in the heads of wild-type and ash1a-morphant embryos. The black arrowhead indicates the nucleus of the posterior commissure and the white arrow indicates the adenohypophysis (G); both are sites where isl1 expression is disrupted in the ash1a morphant (H). A reduction of the number of neurones was observed in the epiphysis of the ash1a morphant (H). By contrast, structures in which ash1a is not expressed, like the trigeminal ganglia, are not affected in the ash1a morphant. (I,J) Expression of ngn1 in wild-type and ash1a-morphant embryos at the 20s stage. (K,L) Expression of ash1a in wild-type and ngn1-morphant embryos at the 17s stage. Hy, hypothalamus; Tg, trigeminal ganglia. Scale bars: in A, 10 µm for A-F,I-L; in H, 50 µm for G,H.

View larger version (61K): [in a new window]   Fig. 4. Ash1a and Ngn1 function downstream of Flh and upstream of neuroD. Dorsal views of brains with anterior at the top, showing expression of flh and neuroD in wild-type (WT), ash1a, ngn1 and ash1a/ngn1-double morphant embryos. Probe and stage are indicated on the left, `genotype' above. Expression of flh was unaffected following the impairment of either or both Ash1a and Ngn1 function (B-D). By contrast, expression of neuroD was affected by the reduction of Ash1a (F) or of both Ash1a and Ngn1 (H). Scale bar: 10 µm.

View larger version (129K): [in a new window]   Fig. 5. Ash1a and Ngn1 regulate the expression of Notch ligands. Dorsal views of brains showing expression of deltaA, deltaB and deltaD in wild-type (WT), flh–/–, and single ash1a, ngn1 or double morphants. Probes used are indicated bottom right, stage bottom left, and `genotype' at the top right of each panel. Arrowheads in B indicate two faint deltaA-positive cells. Expression of all three Delta genes was affected in the ash1a morphants (C,F,I,K,O,S), and more severely reduced or completely absent in the ash1a/ngn1-double morphants (M,Q,U). Scale bar: 10 µm.

View larger version (75K): [in a new window]   Fig. 6. Flh regulates aspects of epiphysial development independent of Ash1a and Ngn1. Dorsal views of brain showing expression of otx5, tbxC and flh in wild-type (WT), flh, and single ash1a, ngn1 or double morphants. Probes used are indicated on the bottom right, stage on the bottom left and `genotype' at the top right of each panel. Ash1a and Ngn1 are implicated in the regulation of otx5 (B,D) but not of tbxC (G,I), whereas Flh is required for the expression of both genes (E,J). In addition, Flh is involved in the regulation of its own expression (N). Scale bar: 10 µm.

View larger version (63K): [in a new window]   Fig. 7. Impairment of Ash1a and of Ngn1 activity affects both photoreceptors and projection neurones. Dorsal views of brains showing expression of onecut and opsin in wild-type (WT) and single ash1a, ngn1 or double-morphant embryos. Probe and stage are indicated on the left and `genotype' above. Both markers were affected following the impairment of Ash1a (B,D,H) or of both Ash1a and Ngn1 activity (F,J). Scale bar: 15 µm.

View larger version (59K): [in a new window]   Fig. 8. Spatially distinct populations of epiphysial neurones show different requirements for flh, ash1a and ngn1. Dorsal (A-D) and lateral (E-G) views of brains with anterior at the top (A-D) or to the right (E-G), showing expression of isl1 at 24 hours in wild-type (WT), flh-mutant and ash1a-morphant embryos, and a flh mutant injected with ash1a MO. In E-G, black arrowheads indicate the limits of the epiphysis, which is marked by a line; white arrowheads indicate the nucleus of the posterior commissure. In the ash1a morphant, remaining neurones are located posteriorly (F). The injection of ash1a MO into the flh mutant leads to the loss of the remaining isl1-positive neurones (D). Scale bar: 10 µm.

View larger version (11K): [in a new window]   Fig. 9. Proposed interactions between Flh, Ash1a, Ngn1 and other regulators during epiphysial neurogenesis. Flh has several distinct activities, it regulates its own expression (green solid arrow), and the expression of the bHLH transcription factors Ash1a and Ngn1 (in purple), which in turn activate lateral inhibition genes (in red), and the potential differentiation factors NeuroD, Otx5 and Onecut. Flh also activates TbxC expression independently of Ash1a and Ngn1 activity; the function of TbxC in the epiphysis is currently unknown (blue dashed arrow with a question mark). In addition, roles for Flh in the transcriptional regulation of lateral inhibition and differentiation genes, independent of the regulation of ash1a and ngn1 transcription, is inferred from analysis of the epiphysis in flh mutants (green dashed arrows with question marks, see text for details). Note that the early expression of ash1a is independent of Flh suggesting that another (as yet unknown) factor operates, possibly redundantly with Flh, to regulate bHLH gene expression at early stages (see Discussion).