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Introduction 

Asymmetric cell division is the process by which a mother cell divides to generate two daughter cells that adopt distinct cell fates.  This process is essential to generate cellular diversity in multicellular organisms.  Both cell signaling and the asymmetric segregation of intracellular proteins are likely required to specify distinct daughter cell fates during asymmetric cell division. Do all organisms use similar signaling pathways?  Are asymmetrically localized determinants conserved between organisms or cell types?  How does an asymmetrically localized factor ultimately lead to differential gene expression necessary for cell fate determination?

To address these questions, the Hawkins’ lab utilizes the nematode Caenorhabditis elegansC. elegans is ideally suited to investigate the mechanisms controlling asymmetric cell division.  In the hermaphrodite, all 302 neurons arise from asymmetric cell divisions.  Since the entire cell lineage is known, the timing, location and polarity of cell divisions can be analyzed at the resolution of single cells.  In addition, a wealth of molecular and genetic tools also makes C. elegans a powerful system in which to identify and analyze genes involved in asymmetric cell division.

The Hawkins’s lab studies how the asymmetrically localized protein HAM-1 and the highly conserved Wnt signaling pathway regulate asymmetric cell division. 

Wnt signaling/DSH-2 and asymmetric cell division

Wnt signaling regulates many asymmetric cell divisions in C. elegans.  We have focused on the role of a C. elegans Dishevelled homolog, DSH-2, and have uncovered several roles for DSH-2 during development. DSH-2 is required for asymmetric neuroblast division during embryogenesis and thus is required for the proper generation of many neurons. In addition, DSH-2 also functions in non-neuronal lineages to regulate asymmetric cell division including the somatic gonadal precursor cells, Z1 and Z4.  Finally, complete loss of DSH-2 function also results in embryonic lethality due to defects in ventral enclosure.

In wild type embryos, DSH-2 is localized to the cell cortex.  In mom-5/Fz mutant embryos, DSH-2 is delocalized to the cytoplasm.

To identify genes functioning with DSH-2 in asymmetric neuroblast division, we preformed a genetic suppressor screen.  This screen was highly successful and we isolated over 60 suppressors, all of which are dominant.  These suppressor mutations could be activating mutations in downstream pathway components or in parallel signaling pathways that function in concert with DSH-2 to regulate asymmetric cell division.  We have focused our characterization on three of the strongest suppressors, Sup245, Sup305 and Sup327.  All three suppressors suppress asymmetric neuroblast division defects, embryonic lethality and Z1/Z4 asymmetric division defects. Genetic mapping experiments placed Sup305 and Sup327 in the same 800 kb interval on the left arm of chromosome I and Sup245 tightly linked to unc-54 on the right arm. There are no known Wnt signaling pathway genes in these regions and thus we have potentially identified novel genes that function with DSH-2. To determine the molecular nature of the suppressor mutations sent the suppressor strains for whole genome sequencing and are currently analyzing the sequences. 

To understand how signaling through DSH-2 regulates asymmetric neuroblast division, we analyzed the requirements for different DSH-2 domains. Dsh is a key component of at least three distinct Wnt signaling pathways: (i) the Wnt/β-catenin pathway; (ii) the Wnt/Planar Cell Polarity (PCP) pathway; and (iii) a Wnt/Ca2+ pathway. All Dsh proteins contain three highly conserved domains; an N-terminal DIX domain, a central PDZ domain and a C-terminal DEP domain.  The DIX domain is essential for Wnt/β-catenin signaling, while the DEP domain is required for β-catenin independent signaling. Our domain analysis suggested that DSH-2 functions in a β-catenin independent Wnt pathway for asymmetric neuroblast division. However, more than one pathway downstream of DSH-2 may function in asymmetric division of Z1 and Z4.  We have identified a Frizzled receptor, MOM-5, that functions upstream of DSH-2 in asymmetric neuroblast division and have demonstrated a role for a Wnt, CWN-1.      

  1. A. A single CAN neuron in a wild type animal.

  2. B.A duplicated CAN neuron in a dsh-2 mutant

HAM-1; an asymmetrically localized transcription factor

HAM-1 is a 414 amino acid that is asymmetrically localized at the cell cortex during embryogenesis and required for many asymmetric neuroblast divisions.  The N-terminus of HAM-1 contains a DNA binding winged helix domain, and is 33% identical to the human transcription factor STOX1. At first, the significance of this homology was unclear.  Using anti-HAM-1 specific antibodies, HAM-1 was observed by immunofluorescence microscopy to be at the cell cortex, and not in the nucleus as predicted if HAM-1 were a DNA-binding transcription factor.  However, when we fused GFP to the N-terminus of HAM-1, transgenic embryos expressing the GFP::HAM-1 fusion protein exhibited strong GFP fluorescence in the nucleus, in addition to the cell cortex. 

  1. A. An embryo expressing  GFP::HAM-1 stained with anti-GFP antibodies                                    

  2. B. A GFP:HAM-1 embryo analyzed directly for GFP fluorescence

What is the relationship between HAM-1 at the cell cortex and in the nucleus?  What is the mechanism by which HAM-1 becomes asymmetrically localized?  To address these questions, we plan to visualize the dynamics of HAM-1 in dividing cells by 4D microscopy,  What genes does HAM-1 transcriptionally regulate to control asymmetric cell division? Chromatin immunoprecipitation (ChIP), followed by Illumina sequencing will be a key approach to define a transcriptional program necessary for asymmetric neuroblast division. 

From a combination of HAM-1 deletion analysis and site-directed mutagenesis, we determined that at least two regions of the protein are necessary to mediate cortical localization; the very N-terminus and an SH3-binding motif in the middle of the protein. In contrast we identified two nuclear localization sequences (NLSs) within the C-terminal half of HAM-1. Mutation of both NLSs eliminated nuclear localization, and disrupted ham-1 function. We also showed that nuclear export is regulated by the nuclear export receptor CRM-1. Finally, to determine if HAM-1 functions in the nucleus, we fused an SV40 NLS to the N-terminus of GFP::HAM-1 after disrupting its endogenous NLSs. The SV40 NLS restored both the nuclear localization of the mutant GFP::HAM-1 and the ability of the fusion protein to rescue ham-1 mutant defects. These results strongly suggest a nuclear role for HAM-1. We propose that the asymmetric localization of HAM-1 at the cell cortex is a mechanism to distribute the protein between daughter cells to mediate a differential transcription program. 

C.  An embryo expressing GFP fused to the C-terminal half of HAM-1 containing both NLSs.

D.  Expression of GFP::HAM-1 lacking both

NLSs.