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First published online 14 June 2006
doi: 10.1242/dev.02447

Development 133, 2747-2756 (2006)
Published by The Company of Biologists 2006
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The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions

Shaun Cordes1, C. Andrew Frank1,* and Gian Garriga1,2,{dagger}

1 Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
2 Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.


Figure 1
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  		Fig. 1. pig-1 mutants have extra neurons derived from multiple lineages. (A) Schematic diagram of an L1 hermaphrodite: anterior to the left, dorsal up. Neurons produced from lineages affected in pig-1 mutants are indicated. (B) Lineages affected in pig-1 mutants. NB, neuroblast. PQR, PVM and SDQL are produced by the left Q neuroblast, and AQR, AVM and SDQR are produced by the right Q neuroblast. Note that the PVQ lineage is not affected in pig-1 mutants but is shown to indicate its relationship to the HSN/PHB lineage. (C) Lineages unaffected in pig-1 mutants. Neurons that were examined are shaded black. DD1,3,5 are produced on the left side of the body, and DD2,4,6 on the right. (B,C) Neurons were identified as described in the Materials and methods. Cells that were not scored are unshaded. (D-H) pig-1 mutants have extra neurons derived from the HSN/PHB neuroblast lineage. (D) Schematic diagram of the wild-type HSN/PHB neuroblast lineage. (E,F) Images of wild-type (E) and pig-1(gm301) (F) hermaphrodites stained with an anti-serotonin antiserum to visualize the HSNs (arrows). (G,H) Composite fluorescence and DIC images of wild-type (G) and pig-1(gm301) (H) hermaphrodites expressing the PHB-specific reporter gmIs22 [nlp-1::gfp] (arrows). Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2. pig-1 ced-3 and ham-1 HSN and PHB phenotypes. (A) pig-1 mutants have extra phasmid (PHA or PHB) neurons and extra HSNs. Mutations in pig-1 interact synergistically with mutations in ced-3 to control the penetrance of extra neurons. The number of PHA/Bs was scored using the integrated reporter gmIs12 [srb-6::gfp], and the number of HSNs was scored by immunostaining with an anti-serotonin antiserum. (B) The extra phasmid neurons in pig-1 mutants are PHBs. The numbers of PHA and PHB neurons were determined using the reporters gmIs22[nlp-1::gfp] and ynIs45[flp-15::gfp], respectively. (C) Mutations in pig-1 are epistatic to ham-1. (D) pig-1 is epistatic to ham-1 in a ced-3 background. (C,D) The numbers of HSNs and PHBs were scored as described in A. For all genotypes, the number of lineages scored is shown below the histogram in parentheses.

 

Figure 3
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  		Fig. 3. pig-1 regulates additional neuroblast lineages. (A-D) Neurons were identified as described in the Materials and methods. The number of lineages scored per genotype is shown in parentheses; ND, not determined. (A) pig-1 mutants have extra AVM, PVM and SDQ neurons, all descendants of the Q.p neuroblast. (B) pig-1 mutants have extra AQR and PQR neurons, and extra M4 neurons. (C) pig-1 mutants have extra I2 neurons. The average number of I2 neurons per lineage is shown above the bars of the histogram. (D) pig-1 mutants have extra PLMs and mutations in pig-1 are epistatic to mutations in ham-1. As in the HSN/PHB lineage, mutations in pig-1 interact synergistically with mutations in the pro-apoptotic genes ced-3 and egl-1 to control the penetrance of extra neurons in the Q.p (A), I2 (C) and PLM/ALN (D) lineages. (We did not examine pig-1 interactions with ced-3 or egl-1 in the lineages that produce the A/PQR and M4 neurons.)

 

Figure 4
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  		Fig. 4. pig-1 regulates the positioning of the cleavage plane in the HSB/PHB and Q.p neuroblasts. (A-E) pig-1 disrupts the size asymmetry of the daughters of the HSN/PHB neuroblast. The nuclear-localized reporter gmIs20 (hlh-14::gfp) was used to identify the daughters of the HSN/PHB neuroblast and measure the size of their nuclei (Frank et al., 2005Go). All images show a ventral view of a single embryo with anterior to the left. In each image, the anterior (arrow) and posterior (arrowhead) daughters of the HSN/PHB neuroblast are magnified in the inset. (A) Wild type, (B) pig-1(gm301), (C) ham-1(gm279) and (D) pig-1(gm301) ham-1(gm279). Scale bars: 5 µm. (E) Quantification of the ratio in nuclear diameter between the anterior and posterior daughters of the HSN/PHB neuroblast in multiple genotypes; n, number of pairs of neuroblast daughters scored. (F) Quantification of the ratio in cell size between the posterior and anterior daughters of the Q.p neuroblast; n, number of pairs of neuroblast daughters scored. The transcriptional reporter ayIs9 (Pegl-17::gfp) was used to identify and measure the size of the daughters of the Q.p neuroblast. Like the HSN/PHB neuroblast division, the Q.p neuroblast division is more symmetric in pig-1 mutants.

 

Figure 5
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  		Fig. 5. Molecular characterization of pig-1. (A) Schematic diagram of the pig-1 transcript. White boxes represent exons, and the location of the SL1-splice leader is indicated. The terminal intron of pig-1 contains two genes, depicted as grey arrows, that are transcribed in the opposite orientation to pig-1. (B) Schematic diagram of the domain architecture of the PIG-1 protein, depicting the N-terminal kinase domain (unshaded) and the C-terminal kinase-associated domain (black). The locations and identities of the gm300, gm301 and gm280 missense mutations are shown above the diagram. Below the diagram is a table comparing each domain in PIG-1 to orthologs in frog, fish, mouse and human. (C) Phylogenetic relationship of PIG-1 to selected members of the PAR-1/SAD-1/Kin1 superfamily of serine/threonine kinases. PIG-1 clusters with other MELK family members.

 

Figure 6
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  		Fig. 6. Expression and subcellular localization of PIG-1. (A-F) Images of larvae expressing Ppig-1::gfp: anterior, left; ventral, up. (A,C,E) DIC microscopy; (B,D,F) GFP fluorescence. (A,B) L1 larva expressing Ppig-1::gfp in the Q neuroblast (arrow) and V5 seam cell (arrowhead). (C,D) L1 larva expressing Ppig-1::gfp in the Q neuroblast daughters Q.a (arrow) and Q.p (arrowhead). (E,F) L1 larva expressing Ppig-1::gfp in the Q lineage descendants AQR (asterisk), AVM (arrow), and SQDR (arrowhead). (G,H) DIC (G) and fluorescence (H) images of an embryo expressing Ppig-1::pig-1::gfp. pig-1::gfp is present throughout the cytoplasm and excluded from the nucleus. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. PIG-1 acts cell-autonomously to regulate the asymmetric division of the Q.p neuroblast. The numbers of AVM and PVM neurons were scored using zdIs5 [mec-4::gfp]; the number of lineages scored is shown in parentheses. Expression of pig-1 from the mab-5 promoter does not rescue the extra AVM phenotype of pig-1(gm344) but does rescue the extra PVM phenotype. Statistical analysis was performed using a one-tailed two-sample Z-test (using the normal approximation to the binomial distribution) by the StatCrunch program (www.statcrunch.com).

 

Figure 8
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  		Fig. 8. Modeling the HSN/PHB lineage in pig-1 and ham-1 mutants. In wild-type animals, the HSN/PHB neuroblast is polarized along the AP axis, with an anteriorly displaced cleavage plane (vertical dotted line) and posteriorly localized determinants of neural precursor fate (gray circles). The neuroblast divides to produce a small anterior daughter that inherits no determinants and undergoes apoptosis, and a larger posterior daughter that inherits the determinants and becomes a neural precursor. In pig-1 mutants, the neuroblast does not polarize and this results in a symmetrically localized cleavage plane and uniformly distributed determinants. Both neuroblast daughters inherit these determinants resulting in the production of two neural precursors. In ham-1 mutants, neuroblast polarity is partially inverted along the AP axis resulting in a posteriorly displaced cleavage plane and an anteriorly enriched distribution of cell fate determinants. Extra neurons are produced when a sufficient concentration of determinants enters both neuroblast daughters, resulting in the production of two precursors. No neurons are produced when the anterior daughter undergoes apoptosis and the posterior daughter receives an insufficient concentration of determinants to develop as a precursor. The concentration of determinants in the posterior daughter could fall below a threshold required for precursor fate, either because of stochastic differences in cleavage position or determinant distribution. Neuroblasts in pig-1 ham-1 double mutants are defective for polarity, resulting in pig-1 phenotypes and pig-1 epistasis to ham-1.

 

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© The Company of Biologists Ltd 2006