C. Andrew Frank

University of California, San Francisco; San Francisco CA, United States of America

C Andrew Frank et al. (1999) International C. elegans Meeting "A screen for mutants with defects in asymmetric cell division"

C Andrew Frank, Gian Garriga, Nancy Hawkins

[International C. elegans Meeting, 1999]

The C. elegans protein HAM-1 participates in the asymmetric divisions of at least five neuroblasts. In particular, we are studying its role in the asymmetric division of the HSN/PHB neuroblast, which divides to produce a daughter cell fated to die and the HSN/PHB precursor cell. HAM-1 is asymmetrically distributed in this neuroblast and inherited by the HSN/PHB precursor. Loss-of-function ham-1 mutants can cause the daughter cell that normally does not inherit HAM-1 to assume an HSN/PHB-like fate instead of a cell death fate. This can result in duplications of the HSN and PHB neurons. Since the cell that does not inherit HAM-1 is the cell whose fate is altered by mutations, our current model is that HAM-1 acts as a tether for cell fate determinants rather than as a cell fate determinant itself. We have conducted genetic screens to identify genes that participate in the same process as HAM-1. To perform these screens, we have utilized the gmIs12 strain, which carries an integrated srb-6-gfp fusion, causing GFP to be expressed in several cells, including the PHA and PHB phasmid neurons. We mutagenized this strain and simply looked for abnormal phasmid neuron phenotypes in the F2 generation. From a non-clonal screen of approximately 18,000 genomes, we identified three mutants with the ham-1 phenotype of extra phasmid neurons: ham-1 ( gm214 ), egl-5 ( gm224 ), and lin-32 ( gm239 ). As these mutations were in genes already implicated in HSN/PHB development, we elected to screen further. We next attempted an F2 clonal screen. From a screen of approximately 2600 genomes, we have identified four mutants with extra phasmid neurons, and these mutants still require further characterization. The mutants that produce extra phasmid neurons are most relevant to our study of HAM-1, but in addition, we have identified other mutants as well. A few of our mutants occasionally produce fewer than normal phasmid neurons. One can hypothesize that mutations resulting in fewer phasmid neurons affect genes that may be involved in determining the HSN/PHB precursor fate. We have also found some mutants with abnormal phasmid neuron axon pathfinding.

C Andrew Frank et al. (2002) West Coast Worm Meeting "The Roles of ham-1 and hlh-14 in Neuroblast Divisions"

Nancy Hawkins, Gian Garriga, C Andrew Frank

[West Coast Worm Meeting, 2002]

Asymmetric cell division is a primary mechanism for generating neuronal diversity in C. elegans. The HAM-1 protein is asymmetrically distributed in many mitotic embryonic cells and participates in the divisions of several different neuroblasts. In the HSN/PHB lineage, for example, the HSN/PHB neuroblast (ABpl/rapppap) divides asymmetrically. In wild type, its anterior daughter dies and its posterior daughter becomes the HSN/PHB precursor. However, in loss-of-function ham-1 mutants, both daughters can adopt an HSN/PHB precursor-like fate resulting in extra HSN and PHB neurons. This phenotype is incompletely penetrant; often the daughter cell fated to die will still die in ham-1 mutants, but aspects of its death may be abnormal such as delayed onset or inappropriate corpse persistence.

C Andrew Frank et al. (2001) International C. elegans Meeting "Genes that Affect Asymmetric Cell Division and Cell Fate in the Nervous System"

Gian Garriga, C Andrew Frank

[International C. elegans Meeting, 2001]

The C. elegans protein HAM-1 is asymmetrically distributed in several cells in the dividing embryo and participates in the asymmetric divisions of at least five neuroblasts. In particular, we study the role of HAM-1 in the division of the HSN/PHB neuroblast. Antibody staining analyses show that HAM-1 is asymmetrically distributed in this neuroblast and inherited by its posterior daughter, the HSN/PHB precursor. Loss of ham-1 function can cause the daughter cell that normally does not inherit HAM-1 to transform into an extra HSN/PHB precursor. This transformation results in duplications of the HSN and PHB neurons. Our current model is that HAM-1 acts as a tether for cell fate determinants and restricts the localization of these determinants to the HSN/PHB precursor. With the overall goal of identifying other players in this process, we conducted three genetic screens for mutants with Ham-1 phenotypes. Each screen was done in a gmIs12 background, a strain that expresses GFP in the PHB phasmid neuron, one of the cells duplicated in ham-1 mutants. For each screen, we simply looked for mutants with abnormal numbers of GFP-expressing phasmid neurons. We isolated two main classes of mutants. The first class has extra phasmid neurons; we found 16 of these mutants, at least nine of which are in genes previously characterized: ham-1 ( gm214 , gm267 , and gm279 ), ced-3 ( gm265 and gm266 ), egl-5 ( gm224 and gm316 ), lin-32 ( gm239 ), and egl-27 ( gm314 ). Three more mutants in this class are allelic: gm280 , gm300 , and gm301 . The second class of mutants has fewer than normal phasmid neurons; we found 16 of these mutants. This class of mutants is interesting because this is the phenotype one would expect for the loss of a determinant that specifies HSN/PHB precursor fate. We were intrigued to find that egl-27 mutants have extra phasmid neurons. EGL-27 is a homolog of MTA1, a member of the NURD complex (nucleosome remodeling and histone deacetylase). egl-27 ; gmIs12 worms have extra phasmid neurons infrequently, but egl-27 ; gmIs12 ; ham-1 ( n1811 or gm267 ) animals have a significantly enhanced PHB duplication defect. egl-27 has previously been implicated as a possible transducer of Wnt signaling, a process known to affect cell polarity, and experiments are underway to explore what roles EGL-27 and other members of the NURD complex might be playing in the HSN/PHB lineage in conjunction with HAM-1. Another mutant we have been characterizing that affects this process is gm34 . gm34 mutants are frequently missing HSNs and PHBs, and this phenotype is epistatic to ham-1 , ced-3 , and lin-32 mutations. We cloned the defective gene by conventional mapping and injection rescue; its sequence (C18A3.8) predicts that it is a basic helix-loop-helix (bHLH) transcription factor. LIN-32 is another bHLH that plays a role in the HSN/PHB lineage, and since bHLH proteins are known to regulate transcription of target genes as dimers, we are currently exploring the possibility that this new bHLH and LIN-32 interact to determine the fates of cells in the HSN/PHB neuroblast lineage.

C Andrew Frank et al. (2003) International Worm Meeting "HAM-1 may influence spindle position in asymmetrically dividing neuroblasts"

C Andrew Frank, Gian Garriga, Nancy Hawkins

[International Worm Meeting, 2003]

Asymmetric cell division is a primary mechanism for generating neuronal diversity in C. elegans. A novel protein, HAM-1, is asymmetrically distributed in many mitotic embryonic cells and participates in the divisions of several neuroblasts. For example, the HSN/PHB neuroblast (ABpl/rapppap) divides asymmetrically. In wild type, its anterior daughter dies and its posterior daughter becomes the HSN/PHB precursor. However, in loss-of-function ham-1 mutants, both daughters can adopt an HSN/PHB precursor-like fate resulting in extra HSN and PHB neurons. This phenotype is incompletely penetrant; often the daughter cell fated to die will still die in ham-1 mutants, but aspects of its death may be abnormal such as delayed onset or inappropriate corpse persistence. With the overall goal of identifying additional components involved in the proper execution of the HSN/PHB lineage, we conducted genetic screens for mutants with extra PHBs. New alleles of ham-1 were isolated in these screens, and subsequent analysis of all available alleles has helped elucidate the role HAM-1 plays in asymmetric cell division. We find that HAM-1 can interact with itself in the yeast two-hybrid system, but the missense mutation ham-1(n1811) disrupts this interaction and the ability of the HAM-1 protein to localize at the cell periphery. Together, these data suggest that HAM-1 multimerization may be necessary for its proper localization. Furthermore, lineage analysis using the nonsense mutant, ham-1(gm279), has allowed us to identify additional defects in the HSN/PHB neuroblast division. The daughter cells of this division often end up oriented abnormally along the A/P axis in ham-1(gm279) embryos. When the anterior daughter does die, its corpse is aberrantly large. Together, these data suggest that HAM-1 may control the positioning of the HSN/PHB neuroblast spindle. We also note that many of the other corpses in ham-1(gm279) embryos are unusually large, suggesting that ham-1 plays a major role in asymmetric cell division at this stage of development. Our screens have also identified new alleles of ced-3 (caspase), egl-5 (homeodomain), egl-27 (MTA1/NURD complex), lin-32 (bHLH protein), and hlh-14 (bHLH protein). All of these genes play roles in the proper execution of the HSN/PHB lineage. We have performed a number of follow-up experiments to pinpoint the functions of these genes in neuroblast divisions.

C Andrew Frank et al. (2003) International Worm Meeting "HLH-14 is an Achaete-Scute-like basic helix-loop-helix factor necessary for the execution of specific neuronal fates"

C Andrew Frank, Gian Garriga

[International Worm Meeting, 2003]

Neural basic helix-loop-helix (bHLH) proteins act at several stages of neuronal development, including the early specification of neuroblast lineages and the late differentiation of postmitotic neurons. We report the characterization of hlh-14, which encodes a C. elegans Achaete-Scute-like bHLH protein. A number of neuroblasts express HLH-14, including the neuroblast that produces the PVQ, HSN, and PHB neurons. hlh-14 mutants lack all three of these neurons. Sometimes this neuron loss is accompanied by inappropriate duplications of a closely related hypodermal cell, the T cell. We hypothesize a cell fate transformation of the PVQ/HSN/PHB neuroblast into its sister cell, the hyp7/T blast cell, is at least partially responsible for these phenotypes. Lineage analysis of hlh-14 mutant embryos is consistent with this model. We find that misexpression of HLH-14 off of a heat shock promoter can generate extra neurons, supporting the idea that HLH-14 may be a proneural bHLH factor. We also find that hypomorphic mutations in hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, can exacerbate hlh-14 neuron loss, indicating that HLH-2 may interact with HLH-14 in neuronal development. We propose that HLH-14 acts in specific C. elegans neuroblast lineages to determine neuronal fate.

CF2218

Caenorhabditis elegans

DR441

Caenorhabditis elegans

Fire A et al. (1994) Worm Breeder's Gazette "Vectorology I: New lacZ Vectors (Building a Better Gene Trap)."

Fire A, Xu SQ

[Worm Breeder's Gazette, 1994]

Vectorology I: New lacZ vectors ("building a better gene trap") Andrew Fire and SiQun Xu Carnegie Institution of Washington, Baltimore, Md 21210

PHX3634

Caenorhabditis elegans