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First published online 19 November 2003
doi: 10.1242/dev.00894

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HLH-14 is a C. elegans Achaete-Scute protein that promotes neurogenesis through asymmetric cell division

C. Andrew Frank, Paul D. Baum^* and Gian Garriga

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3204, USA

View larger version (105K): [in a new window]   Fig. 1. Grossly visible phenotypes of hlh-14 mutants. (A) The tail of a wild-type C. elegans larva. (B) An hlh-14(gm34) larva. hlh-14 mutant worms can have disorganized, lumpy tails (arrow) and, less often, lumps at other positions along the AP axis. The cellular bases for these morphological defects are unclear. (C,D) hlh-14(RNAi) larvae. hlh-14(RNAi) larvae can display a range of morphological defects. (C) A severely affected hlh-14(RNAi) animal with a large posterior bulge (arrow). (D) An animal mildly affected by hlh-14(RNAi) treatment, showing only a small bump in the tail (arrow). Some hlh-14(RNAi) animals have a normal body structure, but still display neuronal phenotypes, indicating that these two phenotypes can be uncoupled.

View larger version (39K): [in a new window]   Fig. 2. hlh-14 gene structure, predicted protein sequence and comparison with other A S family members. (A) Schematic representation of hlh-14 mRNA. By 5' and 3' RACE, we detected three different hlh-14 cDNAs, predicting three different forms of HLH-14 protein (Materials and methods). Analyses of these cDNA species indicate that hlh-14 mRNA is not trans-spliced. The three forms of mature hlh-14 mRNA have unique start codons (AUG) but encode the same bHLH domain and the same novel C terminus. The shortest form encodes a 148 amino acid protein. The 5' end of the corresponding short cDNA is the only 5' end we could amplify out of a RACE cDNA library without performing a second, nested PCR reamplification (Materials and methods). Therefore, it is likely that this form is the most abundant form of hlh-14 mRNA in C. elegans. In the corresponding protein, there are only eight amino acids N-terminal of the bHLH domain (MAKKNQVA). (B) Primary amino acid sequences of HLH-14. The three different start methionines are enlarged. For the two longer forms of HLH-14, alternative beginning peptides are boxed. The leucine and glutamine residues changed, respectively, by the hlh-14(gm34) missense mutation and the hlh-14(ju243) nonsense mutation are enlarged and underlined. Both lesions are in the conserved bHLH domain. (C) Alignment of the bHLH domains of HLH-14, HLH-3 (C. elegans), Achaete (Drosophila), Scute (Drosophila) and Mash1 (Ascl1; mouse). Residues shaded in black are identical. Lightly shaded residues are similar to corresponding black residues. Consensus residues are present in at least three of the five proteins aligned. The residues affected by the hlh-14(gm34) and hlh-14(ju243) mutations are detailed.

View larger version (126K): [in a new window]   Fig. 3. HLH-14::GFP is expressed in the PVQ/HSN/PHB neuroblasts and their descendants. All images in this series show a ventral view. Anterior is at the upper left corner and posterior is at the lower right corner. Images in this series reveal posterior embryonic HLH-14::GFP expression. Nomarski photomicrographs (A,C,E,G,I,K) and the corresponding fluorescence photomicrographs (B,D,F,H,J,L) of wild-type embryos containing a gmIs20[hlh-14::gfp] transgene. (A,B) HLH-14::GFP is expressed in the right PVQ/HSN/PHB neuroblast, ABprapppa (arrow). (C,D) GFP expression of the same embryo shown in A and B, showing a different focal plane. HLH-14::GFP is expressed in the left PVQ/HSN/PHB neuroblast, ABplapppa (arrow). (E,F) HLH-14::GFP is expressed in the daughter cells of the PVQ/HSN/PHB neuroblasts: the anterior PVQ neuroblasts (open arrows) and the posterior HSN/PHB neuroblasts (closed arrows). We have tentatively identified the central GFP-expressing cell as the neuroblast C.aapa (arrowhead). (G,H) HLH-14::GFP is expressed in the anterior daughter of the right HSN/PHB neuroblast, a cell fated to die (closed arrow), and in its larger sister cell, the right HSN/PHB precursor (open arrow). The asterisk indicates the right PVQ neuroblast, which is out of focus. (I,J) HLH-14::GFP is expressed in the daughters of the left HSN/PHB neuroblast (arrows analogous to H). (K,L) HLH-14::GFP is expressed in the daughters of the PVQ neuroblast, the anterior PVQ neurons (closed arrows) and their posterior sisters, which are fated to die (open arrows). Scale bar: 5 µm. (M) Cell lineage depicting the PVQ/HSN/PHB neuroblast and its descendants.

View larger version (72K): [in a new window]   Fig. 4. hlh-14 mutants are missing HSN, PHB, and PVQ neurons. Images in this series show lateral views of either wild-type (A,C,E) or hlh-14 mutant (B,D,F) larvae expressing GFP reporters to visualize the HSN, PHB or PVQ neurons. (A,B) Anterior is towards the left and ventral is upwards. (A) GFP expression in an hlh-14(gm34)/mIn1; kyIs179[unc-86::gfp] larva. As this worm is only heterozygous for the hlh-14(gm34) mutation, it appears wild type and expresses GFP in the HSN neuron visible in this focal plane (arrow). The GFP expression in the pharynx of this worm indicates the presence of the mIn1 balancer, and hence, a wild-type copy of the hlh-14 gene. (B) GFP expression in an hlh-14(gm34); kyIs179 larva. No HSNs are visible by GFP expression. The arrow indicates the position where the HSNs would be in a wild-type animal. (C) GFP expression in the tail of a wild-type gmIs12[srb-6::gfp] larva. GFP is visible in both phasmid neurons, PHA and PHB (two arrows), sensory neurons in the tail. (D) GFP expression in an hlh-14; gmIs12 larva. Only one phasmid neuron (PHA) is visible; PHB is missing. (E) GFP expression in an hlh-14(gm34)/mIn1; kyIs39[sra-6::gfp] larva. As this worm is only heterozygous for the hlh-14(gm34) mutation, it appears wild type and expresses GFP in the PVQ neuron visible in this focal plane (arrow). The GFP expression slightly anterior to PVQ is due to the presence of mIn1. (F) GFP expression in an hlh-14(gm34); kyIs39 larva. No PVQs are visible by GFP expression. The arrow indicates the position of the PVQs in a wild-type animal.

View larger version (51K): [in a new window]   Fig. 5. Cell lineage defects in hlh-14 mutants. (A) In wild-type embryos, the ABpl/rappp cell divides to generate the PVQ/HSN/PHB neuroblast and the hyp7/T blast cell. The PVQ/HSN/PHB neuroblast subsequently undergoes a series of divisions, ultimately generating the PVQ, HSN and PHB neurons. The hyp7/T blast cell divides only once during embryogenesis, generating a hyp7 cell and the T blast cell, a seam cell that divides postembryonically. (B) In hlh-14 mutants, the ABpl/rappp cell divides at the appropriate time. However, the presumptive PVQ/HSN/PHB neuroblast does not undergo a series of divisions to generate the PVQ, HSN and PHB neurons. Instead, it divides just once, like its sister cell, the hyp7/T blast cell. In hlh-14 mutants, it is possible that the PVQ/HSN/PHB neuroblast is transformed into a hyp7/T blast-like cell, a model shown in this lineage. (C) A wild-type L1 larva containing a C50B6.8::gfp reporter construct, which expresses GFP in the ten hypodermal seam cells, H0-H2, V1-V6 and T (Mounsey et al., 2002). (D) An hlh-14 mutant L1 larva expressing the C50B6.8::gfp reporter. In addition to expressing GFP in the ten hypodermal seam cells, this larva expresses GFP in an extra cell in the tail, presumably a T-like cell (arrow). In 38% of the of hlh-14 mutants, we observed this ectopic GFP expression (n=60 sides scored). We never observed this extra cell in wild-type animals bearing C50B6.8::gfp.

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