Hammarlund M et al. (1998) West Coast Worm Meeting "unc-70: A REGULATOR OF SYNAPTIC TRANSMISSION"

Jorgensen EM, Hammarlund M

[West Coast Worm Meeting, 1998]

Synaptic transmission is a complex and highly regulated process.Transmission must be regulated such that a given pre-synaptic potentialevokes an appropriate post-synaptic response. In addition, transmissioncan be modified to strengthen or weaken a particular synapse, and theseprocesses may underlie cognitive processes such as learning and memory.We are interested in how levels of synaptic transmission are establishedand maintained at appropriate levels. We are studying the unc-70 locus,which we have identified as playing a key role in regulating levels ofsynaptic transmission. There are two distinct allelic classes of unc-70 mutations. Therecessive class, which may represent loss or reduction of UNC-70 function,has been classified as early larval lethal. We have found conditions thatallow animals homozygous for these alleles to survive and propagate. Thishas allowed us to examine the phenotype caused by these alleles in adultanimals. Animals homozygous for recessive alleles of unc-70 are severelydumpy, attaining only one-third the length of a wild-type adult. Further,these animals are paralyzed and exhibit decreased neurotransmission, asassayed by aldicarb-resistance. These results suggest that in addition togeneral effects on body morphology, UNC-70 is specifically required at thesynapse for normal neurotransmission. The two dominant alleles of unc-70 behave genetically asgain-of-function mutations. Animals homozygous for dominant unc-70alleles appear to be morphologically normal and are active and healthy.However, they are uncoordinated and adopt a curly posture. We haveassayed neurotransmission in these animals by testing for aldicarbresistance. Animals homozygous for dominant alleles of unc-70 arehypersensitive to aldicarb, suggesting that these mutations cause anincrease in neurotransmission. In addition, overexpression of UNC-70 froma transgenic array generates this same phenotype. These results suggestthat UNC-70 may regulate the amount of neurotransmitter released from thesynapse. unc-70 is on Chromosome V just to the right of dpy-11. We haverescued the recessive unc-70 phenotype by injecting a single cosmid in thisregion. We are now proceeding to identify the open reading frame that corresponds to unc-70. Once we have cloned unc-70, we will sequence mutant alleles as a first step toward understanding how the UNC-70 proteinregulates the level of synaptictransmission.

Hammarlund M et al. (2009) Science "Axon regeneration requires a conserved MAP kinase pathway."

Nix P, Hauth L, Hammarlund M, Bastiani M, Jorgensen EM

[Science, 2009]

Regeneration of injured neurons can restore function, but most neurons regenerate poorly or not at all. The failure to regenerate, in some cases, is due to a lack of activation of cell-intrinsic regeneration pathways. Thus, these pathways might be targeted for the development of therapies that can restore neuron function after injury or disease. Here, we show that the DLK-1 mitogen-activated protein (MAP) kinase pathway is essential for regeneration in Caenorhabditis elegans motor neurons. Loss of this pathway eliminates regeneration, whereas activating it improves regeneration. Further, these proteins also regulate the later step of growth cone migration. We conclude that after axon injury, activation of this MAP kinase cascade is required to switch the mature neuron from an aplastic state to a state capable of growth.

Hammarlund M et al. (2014) Curr Opin Neurobiol "Axon regeneration in C. elegans."

Hammarlund M, Jin Y

[Curr Opin Neurobiol, 2014]

Single axon transection by laser surgery has made Caenorhabditis elegans a new model for axon regeneration. Multiple conserved molecular signaling modules have been discovered through powerful genetic screening. In vivo imaging with single cell and axon resolution has revealed unprecedented cellular dynamics in regenerating axons. Information from C. elegans has greatly expanded our knowledge of the molecular and cellular mechanisms of axon regeneration.

Hammarlund M et al. (2007) PLoS Biol "Open syntaxin docks synaptic vesicles."

Watanabe S, Olsen S, Palfreyman MT, Hammarlund M, Jorgensen EM

[PLoS Biol, 2007]

Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.

Hammarlund M et al. (1997) International C. elegans Meeting "A Genetic Screen For Downstream Effectors of ICE"

Hammarlund M, Maricq AV

[International C. elegans Meeting, 1997]

CED-3 and its human homolog, Interleukin-1-beta Converting Enzyme (ICE), are known to play a central role in a cell's decision to undergo apoptotic death. However, the downstream effectors of these cysteine proteases are not well understood. In an effort to understand the processes by which cysteine protease activation leads to apoptotic death, we are conducting screens for supressors of ICE-induced cell death in C. elegans. In previous work with Cori Bargmann (UCSF), we fused the glr-1 promoter to the human ICE sequence and integrated this construct. glr-1::ICE animals are touch-defective due to the ICE-induced apoptotic death of interneurons. We mutagenized this strain with EMS and screened for suppression of the touch phenotype. Four suppressor strains were isolated from this screen. These strains are currently being characterized. Since the pathway that leads from ICE to apoptosis is likely to be extensively branched, we are taking a parallel approach that will boost our screening efficiency. We obtained the ceh-28 promoter from Brian Harfe and Andrew Fire, who showed that ceh-28::gfp is expressed only in the M4 pharyngeal neuron. Leon Avery has shown by laser ablation that M4 is required for survival beyond L1. M4 innervates the posterior half of the isthmus; when M4 is killed, the isthmus remains closed and the animal starves. Avery also found that M4-ablated animals are viable on media containing arecoline, a drug which depolarizes M4's post-synaptic target. We have fused the ceh-28 promoter to the human ICE sequence. We expect ceh-28::ICE animals to be non-viable on normal media due to apoptotic death of M4, but viable on arecoline. This will allow us to select for suppressors of the M4-ablated phenotype by growing ceh-28::ICE animals on arecoline, mutagenizing, and then moving F2's to normal media. This selection is likely to yield several broad classes of genes. In addition to downstream effectors of ICE, we expect to find mutations that allow ceh-28::ICE animals to survive by depolarizing the isthmus.

Hammarlund M et al. (2007) J Cell Biol "Axons break in animals lacking beta-spectrin."

Jorgensen EM, Hammarlund M, Bastiani MJ

[J Cell Biol, 2007]

Axons and dendrites can withstand acute mechanical strain despite their small diameter. In this study, we demonstrate that beta-spectrin is required for the physical integrity of neuronal processes in the nematode Caenorhabditis elegans. Axons in beta-spectrin mutants spontaneously break. Breakage is caused by acute strain generated by movement because breakage can be prevented by paralyzing the mutant animals. After breaking, the neuron attempts to regenerate by initiating a new growth cone; this second round of axon extension is error prone compared with initial outgrowth. Because spectrin is a major target of calpain proteolysis, it is possible that some neurodegenerative disorders may involve the cleavage of spectrin followed by the breakage of neural processes.

Hammarlund M et al. (2005) Genetics "Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans."

Jorgensen EM, Davis MW, Hammarlund M, Dayton D, Nguyen H

[Genetics, 2005]

The normal distribution of crossovers events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on C. elegans chromosomes, and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase scale gap in sequence homology.

Hammarlund M et al. (2008) J Cell Biol "CAPS and syntaxin dock dense core vesicles to the plasma membrane in neurons."

Hammarlund M, Schuske K, Jorgensen EM, Watanabe S

[J Cell Biol, 2008]

Docking to the plasma membrane prepares vesicles for rapid release. Here, we describe a mechanism for dense core vesicle docking in neurons. In Caenorhabditis elegans motor neurons, dense core vesicles dock at the plasma membrane but are excluded from active zones at synapses. We have found that the calcium-activated protein for secretion (CAPS) protein is required for dense core vesicle docking but not synaptic vesicle docking. In contrast, we see that UNC-13, a docking factor for synaptic vesicles, is not essential for dense core vesicle docking. Both the CAPS and UNC-13 docking pathways converge on syntaxin, a component of the SNARE (soluble N-ethyl-maleimide-sensitive fusion protein attachment receptor) complex. Overexpression of open syntaxin can bypass the requirement for CAPS in dense core vesicle docking. Thus, CAPS likely promotes the open state of syntaxin, which then docks dense core vesicles. CAPS function in dense core vesicle docking parallels UNC-13 in synaptic vesicle docking, which suggests that these related proteins act similarly to promote docking of independent vesicle populations.

Hammarlund M et al. (2018) Neuron "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System."

Miller DM, Hobert O, Hammarlund M, Sestan N

[Neuron, 2018]

Differential gene expression defines individual neuron types and determines how each contributes to circuit physiology and responds to injury and disease. The C.elegans Neuronal Gene Expression Map & Network (CeNGEN) will establish a comprehensive gene expression atlas of an entire nervous system at single-neuron resolution.

Hammarlund, M. et al. (2019) International Worm Meeting "The C. elegans Neuronal Gene Expression map and Network (CeNGEN)."

Hammarlund, M., Sestan, Nenad, Miller, D., Hobert, O.

[International Worm Meeting, 2019]

The fundamental architecture of nervous systems is defined by genetic programming, a feature that is especially apparent in the known wiring diagram and developmental lineage of all neurons in C. elegans. As a first step toward linking cell-specific gene expression with neuronal development and function, the CeNGEN project will profile each of the 118 types of C. elegans neurons1. This goal will be accomplished in two parallel tracks: (1) We are using single-cell RNA-Seq to fingerprint each neuron class and to identify potential neuron subtypes that have previously escaped classification: (2) We are using FACS to isolate each neuron type for whole transcriptome analysis to reveal alternatively spliced transcripts, miRNAs and noncoding RNAs. Initial experiments will focus on L4 larvae. Additional approaches will profile earlier developmental stages and sex-specific gene expression for selected groups of neuron types. As it is validated, data will be deposited at GEO and WormBase for use by the C. elegans community. A complete gene expression map of the entire C. elegans nervous system will provide an unprecedented opportunity to link genetic programs with nervous system structure and function. 1. Hammarlund, M., Hobert, O., 3rd, D. M. M. & Sestan, N. The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System. Neuron 99, 430-433 (2018).