Park M et al. (1995) Worm Breeder's Gazette "CeMEF-2 Is Expressed in Muscle and Nerve."

Krause MW, Park M, Lee-Llacer R, Littlejohn R, Dichoso D

[Worm Breeder's Gazette, 1995]

CeMEF-2 is expressed in muscle and nerve Morgan Park, Daryl Dichoso*, Rey Lee-Llacer, Robert Littlejohn, and Michael Krause. Laboratory of Molecular Biology, NIDDK, NIH. *Dept. of Biology, University of Houston. The vertebrate Mef-2 family of genes encodes sequence specific transcription factors of the MADS box class that are related to the serum response factors (SRFs). Mef-2 genes are expressed in a variety of tissues, however, their expression in muscle has received the most attention. In vertebrates, MEF-2 isoforms play a critical role in activating a variety of muscle specific proteins during skeletal muscle differentiation, including a positive feedback regulation of MyoD family members such as MyoD and myogenin. In addition, certain MEF-2 isoforms are expressed in differentiating cardiac tissue and can activate cardiac-specific genes. In Drosophila, there appears to be only a single Mef-2-like gene that is expressed in precursors of all muscle types, with expression in striated muscle preceding that of the MyoD family member, nautilus; essentially no neuronal expression has been observed in flies. We have cloned a C. elegans member of the Mef-2 family by degenerate RT-PCR. PCR results were previously interpreted to suggest as many as five Mef-2-like genes might exist in the worm. However, genomic and cDNA library screens continue to pull out only a single gene. A careful re-examination of the PCR data has allowed us to eliminate (due to suspected PCR errors or contamination) all but a single product from C. elegans, consistent with a single gene. Genomic Southern blots also suggest a single gene, although there is a faintly hybridizing secondary band that can not be explained by the genomic locus we have cloned. We have looked at a single line of animals transformed with a CeMEF-2::beta-galactosidase reporter construct that includes ~7kb of sequence upstream of the ATG in addition to a large (2337bp) first intron. During embryogenesis, weak expression is seen at the tailbud stage in a few unidentified cells in the head region. Subsequently, other cells begin to express the reporter gene and by the three-fold stage, there is very strong expression in what appears to be all muscle cells (bwm, pharyngeal, others?) and all neurons. The large number of staining cells makes it difficult to determine if all minor muscle cell groups (intestinal, uterine, etc.) are expressing. Expression continues in both muscle and nerve to adulthood. The expression pattern of this mef-2::1acZ construct is similar to what is seen in vertebrates, suggesting that this transcription factor is playing a role in the differentiation of muscle and nerve in C. elegans. If the onset of expression during embryogenesis is correctly reflected by our reporter construct, it is unlikely that CeMEF-2 initiates the commitment of precursor blastomeres to these different cell types (eg. in body wall muscle cells, myosin heavy chain is activated prior to our reporter construct). Instead, CeMEF-2 might interact with other tissue-specific factors to enhance, or maintain, the expression of activated genes and help drive terminal differentiation.

Park M et al. (2018) Curr Biol "Aging: Antagonistic Pleiotropy Supported by Gut Eating."

Park M, Wang MC

[Curr Biol, 2018]

A new study has found that, in the nematode worm Caenorhabditis elegans, biomass conversion from theintestine to yolk, mediated by autophagy and insulin/IGF-1 signaling, promotes reproduction in early life but aging in late life.

Park M et al. (1997) Worm Breeder's Gazette "Isolation and characterization of factors that interact with C. elegans MyoD"

Park M, Krause MW, Littlejohn R

[Worm Breeder's Gazette, 1997]

The MyoD family of transcription factors are involved in the specification and development of vertebrate skeletal muscle. These factors contain a conserved basic helix-loop-helix (bHLH) domain that directs DNA binding and protein dimerization. In vertebrates, the MyoD family members heterodimerize with an ubiquitously expressed E protein to activate muscle specific expression. A single homolog of MyoD (CeMyoD) and E proteins (CeE/DA) has been found in C. elegans. Although CeMyoD is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres, CeE/DA is not expressed in these cells. This result raises the question of whether there are other factors that heterodimerize with CeMyoD to direct proper body wall muscle development. To address this question, we have utilized the yeast two-hybrid system to identify gene products that interact with CeMyoD. We fused the bHLH domain of CeMyoD (aa 150-203) to the binding domain of Gal4 to screen a cDNA library containing fusions to the activation domain of Gal4 (the library was kindly provided by Bob Barstead). We isolated 3 potential interactors after screening 1,000,000 clones from the cDNA library. We have sequenced the inserts from these interacting clones. The following is a summary of each of the interacting clones: 1) pal-1. One of the clones identified in the screen is the homeodomain protein pal-1. It is involved in the development of the somatic daughters of the P lineage; the C+D lineages give rise to body wall muscle. Loss of pal-1 leads to a loss of posterior body wall muscle. The link between pal-1 and CeMyoD is not clear since the expression of pal-1 protein disappears prior to the onset of stable CeMyoD expression in the embryo. Additionally, pal-1 expression appears to be restricted from body wall muscle post-embryonically. (Thanks to Craig Hunter and Lois Edgar for sharing information on pal-1). 2) putative GTP binding protein. The second clone is similar to a putative GTP binding protein identified in C. elegans (CGP-1) and in Ascaris (AGP-1). This clone lies on Linkage Group V in cosmid TO4H1 and matches two clones from the Kohara EST library (yk20h4, yk20g2). We have used the cosmid sequence to design primers to PCR amplify a 2.5 kB portion of the genomic sequence upstream of the coding region of the gene to construct GFP and LacZ reporter constructs. The reporters are expressed in body wall muscle, pharyngeal muscle, intestinal muscle, and anal spincter muscle in adults and larvae. The earliest expression with these constructs is seen at the three-fold stage in the pharyngeal and anal spincter muscles. 3) cyclin D homolog. The sequence from the third clone has matches to three ESTs (yk118d3, CEESF44, CEESH01), but no matches to sequences in the genomic database. The predicted protein sequence is similar to Cyclin D. Cyclin D is a G1-cyclin and is involved in decision to differentiate or to re-enter the cell cycle. It is thought that Cyclin D acts as a growth factor sensor to tell cells to exit G1 and differentiate. In myogenesis, there is evidence that the myoD family members cooperate with G1 cyclins (Rb and Cyclin D) to activate skeletal muscle specific genes and to commit cells to muscle cell development. We have generated his-tagged fusion constructs to purify these proteins for use in generating antibodies, in biochemical studies to look at the interactions of these proteins with CeMyoD, and in band shift assays. We are characterizing the expression of these clones by in situ hybridization and their possible function by antisense RNA injection.

Park M et al. (1995) Worm Breeder's Gazette "Antisense RNA inhibition of zygotic genes expressed in muscles and neurons."

Krause MW, Park M

[Worm Breeder's Gazette, 1995]

Inhibition of gene activity using antisense RNA has been a useful tool in the determination of gene function in both prokaryotic and eukaryotic systems. In C. elegans, antisense RNA mediated gene inhibition has been achieved by introducing a promoter driven expression construct of a target DNA fragment in reverse orientation (e.g. unc-22; Fire et al., Development, 1991, 113: 503-514) and by direct injection of the antisense RNA into the hermaphrodite gonad (e.g. par-1; Guo and Kemphues, Cell, 1995, 81: 611-620). In the latter case, injection of antisense RNA to the maternal effect gene par-1 phenocopied a mutation in par-1. We sought to determine if this technique could be utilized in the study of zygotically expressed genes that act later in development. CeMyoD is a member of the family of myogenic basic helix-loop-helix proteins and is involved in the development of body wall muscle (Krause, Bioessays, 1995, 17: 219-228). CeMyoD is stabily expressed beginning at the ~100 cell stage of embryogenesis. We attempted to phenocopy a null mutation in the gene encoding CeMyoD (cc450; Chen et al., Development, 1994, 120: 1631-1641) by injecting antisense RNA transcribed in vitro from the CeMyoD cDNA using T7 polymerase. The antisense RNA reaction (1.0 mg/ml RNA) was injected into each arm of the hermaphrodite gonad at the L4/early adult stage using standard transformation techniques (Mello et al., EMBO J., 1991, 10: 3959-3970). We placed 5-10 injected worms on seeded NGM plates to recover at 20 C for 1-2 days. In the progeny of injected worms, we observed animals that were phenotypically similar to cc450 as seen by their Lumpy/Dumpy appearance and failure to elongate beyond the 2-fold stage. We also observed three other classes of defects: (1) embryos that had more severe defects in morphogenesis than cc450, (2) embryos that elongated beyond 2-fold that were Lumpy, and (3) those that gave rise to larvae with minor head defects. We are not certain what is the cause of this "allelic series", but it may be a dose effect due to the instability or improper partitioning of the antisense RNA or to the interference with processes which utilize other bHLH proteins. On each plate, ~15% of the progeny exhibited a phenotype in the series of defects. No phenocopy defects were seen in progeny of worms injected with a reaction that contained no cDNA template. We have injected the sense RNA and obtain the same results as the antisense RNA injections; the frequency of the phenocopy is similar to that using the antisense RNA. This sense strand effect is similar to what is seen in the phenocopy of par-1 as well as the experience of others using antisense RNA to phenocopy maternal gene products. We have also attempted to phenocopy using antisense oligos. We saw no effect using single antisense oligos (~0.2 mg/ml) or a mixture of several antisense oligos (mix of three oligos at ~0.06 mg/ml each) to CeMyoD. We have injected single stranded DNA to determine if the formation of hybrid RNA leads to the block to gene activity. We were unable to phenocopy cc450 defects using antisense or sense CeMyoD single stranded DNA (~2 mg/ml ssDNA). We have used this technique in the study two other genes that are expressed in muscle and neurons. CeMEF-2 appears to be expressed in all muscles (pharyngeal, body wall, vulval, and male tail) and neurons. CeE12/Da is expressed in cells within the head, tail, and along the ventral region of the embryo at the 2-fold stage; this expression corresponds to regions of neural differentiation. We have cloned these genes using molecular techniques but we have not isolated mutants. We injected worms with antisense RNA from each of these genes. We observed defects in the progeny of worms injected with these antisense RNAs; these defects were limited to tissues that express these genes. In each case tested, the defects were gene-specific, that is, each sequence gave a unique pattern of defects. We have been able to phenocopy the null mutation in the zygotically expressed gene encoding CeMyoD using its cDNA. We have also generated defects in two other zygotically expressed genes that we have cloned. This suggests that the antisense RNA injection technique may provide another reverse genetic tool for the study of zygotically expressed genes in C. elegans.

Park M et al. (1995) International C. elegans Meeting "IDENTIFYING FACTORS INVOLVED IN BODY WALL MUSCLE DEVELOPMENT."

Littlejohn R, Lee-Llacer R, Park M, Krause MW, Dichoso D

[International C. elegans Meeting, 1995]

The MyoD family of transcription factors are involved in the specification and development of vertebrate skeletal muscle. The single MyoD homolog in C. elegans (CeMyoD) is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres. Null mutations in the gene encoding CeMyoD do not prevent the expression of muscle components but result in disorganized muscle structure and defects in morphogenesis. This genetic evidence suggests that additional factors that might work in concert with CeMyoD are necessary for proper body wall muscle development.

Park M et al. (1996) East Coast Worm Meeting "IDENTIFYING FACTORS THAT INTERACT WITH amide IN BODY WALL MUSCLE DEVELOPMENT"

Littlejohn R, Krause MW, Park M

[East Coast Worm Meeting, 1996]

The MyoD family of transcription factors are involved in the specification and development of vertebrate skeletal muscle. These factors contain a conserved basic helix-loop-helix (bHLH) domain that directs DNA binding and protein dimerization. In vertebrates, the MyoD family members heterodimerize with an ubiquitously expressed E protein to activate muscle specific expression. A single homolog of MyoD (CeMyoD) and E proteins (CeE/DA) has been found in C. elegans . Although CeMyoD is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres, CeE/DA is not expressed in these cells. This result raises the question of whether there are other factors that heterodimerize with CeMyoD to direct proper body wall muscle development. To address this question, we have utilized the yeast two-hybrid system to identify gene products that interact with CeMyoD. We have used a construct containing the bHLH domain of CeMyoD fused to the DNA binding domain of Gal4 to screen a cDNA library containing fusions to the Gal4 activation domain (kindly provided by Bob Barstead). In a screen of 700,000 clones, we identified 3 clones that interact with the CeMyoD bHLH-Gal4 BD fusion. We will report on the characterization of these interacting clones.

Park M et al. (1998) East Coast Worm Meeting "A cyclin D is required for larval, but not embryonic, cell divisions in C. elegans."

Krause MW, Park M

[East Coast Worm Meeting, 1998]

In multicellular organisms, the proper timing of cell divisions requires the coordination of developmental signals and factors that control the cell cycle. Cell cycle progression is controlled by the action of cyclin dependent kinases (CDKs). Several factors have been shown to regulate the activity of CDKs; these include cyclins, cyclin-dependent kinase inhibitors (CKIs), phosphorylation, and degradation. Specific cyclins in association with the appropriate CDK act during different stages of the cell cycle. Mitosis is controlled by the mitotic cyclins A and B, whereas G1 cyclins (D and E) function to control the decision to either enter S-phase or arrest in G1. In C. elegans, the post-embryonic blast cells respond to temporal controls and proliferate at specific times during larval development, indicating that the cell cycle in these cells is under developmental regulation. We have identified a C. elegans homolog of the G1-specific cell cycle regulator cyclin D (cyd-1). We will show that cyd-1 is necessary for the division of the post-embryonic blast cells. No embryonic defects are associated with the loss of this cyclin, suggesting that this cyclin function is specific for post-embryonic development, perhaps due to the lack of a G1 phase in embryonic cell cycles. Our model is that cyd-1 is essential to transduce signals for blast cell to exit G1 arrest throughout post-embryonic development.

Park M et al. (1999) Development "Regulation of postembryonic G(1) cell cycle progression in Caenorhabditis elegans by a cyclin D/CDK-like complex."

Krause MW, Park M

[Development, 1999]

In many organisms, initiation and progression through the G(1) phase of the cell cycle requires the activity of G(1)-specific cyclins (cyclin D and cyclin E) and their associated cyclin-dependent kinases (CDK2, CDK4, CDK6). We show here that the Caenorhabditis elegans genes cyd-1 and cdk-4, encoding proteins similar to cyclin D and its cognate cyclin-dependent kinases, respectively, are necessary for proper division of postembryonic blast cells. Animals deficient for cyd-1 and/or cdk-4 activity have behavioral and developmental defects that result from the inability of the postembryonic blast cells to escape G(1) cell cycle arrest. Moreover, ectopic expression of cyd-1 and cdk-4 in transgenic animals is sufficient to activate a S-phase reporter gene. We observe no embryonic defects associated with depletion of either of these two gene products, suggesting that their essential functions are restricted to postembryonic development. We propose that the cyd-1 and cdk-4 gene products are an integral part of the developmental control of larval cell proliferation through the regulation of G(1) progression.

Park M et al. (2011) Neuron "CYY-1/cyclin Y and CDK-5 differentially regulate synapse elimination and formation for rewiring neural circuits."

Jorgensen EM, Watanabe S, Park M, Ou CY, Shen K, Poon VY

[Neuron, 2011]

The assembly and maturation of neural circuits require a delicate balance between synapse formation and elimination. The cellular and molecular mechanisms that coordinate synaptogenesis and synapse elimination are poorly understood. In C. elegans, DD motoneurons respecify their synaptic connectivity during development by completely eliminating existing synapses and forming new synapses without changing cell morphology. Using loss- and gain-of-function genetic approaches, we demonstrate that CYY-1, a cyclin box-containing protein, drives synapse removal in this process. In addition, cyclin-dependent kinase-5 (CDK-5) facilitates new synapse formation by regulating the transport of synaptic vesicles to the sites of synaptogenesis. Furthermore, we show that coordinated activation of UNC-104/Kinesin3 and Dynein is required for patterning newly formed synapses. During the remodeling process, presynaptic components from eliminated synapses are recycled to new synapses, suggesting that signaling mechanisms and molecular motors link the deconstruction of existing synapses and the assembly of new synapses during structural synaptic plasticity.

Park M et al. (1997) International C. elegans Meeting "A PUTATIVE GTP BINDING PROTEIN AND A D-TYPE CYCLIN HOMOLOG INTERACT WITH THE BASIC HELIX-LOOP-HELIX DOMAIN OF C. ELEGANS MYOD."

Krause MW, Park M

[International C. elegans Meeting, 1997]

The MyoD family of transcription factors is involved in the specification and development of vertebrate skeletal muscle. These factors contain a conserved basic helix-loop-helix (bHLH) domain that directs DNA binding and protein dimerization. These factors have been shown to interact with other cellular components (E proteins and G1 cyclins) to direct muscle development. A single homolog of both MyoD (CeMyoD) and E proteins (CeE/DA) have been found in C. elegans. Although CeMyoD is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres, CeE/DA is not expressed in these cells. We have undertaken a two hybrid screen using the bHLH domain of CeMyoD in order to isolate other factors that are involved in body wall muscle specification. We have focused on two clones that were identified in this two hybrid screen; one interacting clone is similar to a putative GTP binding protein of unknown function found in C. elegans (CGP-1) and in Ascaris (AGP-1). It encodes a protein that contains a potential nucleotide binding domain and a leucine zipper. Reporter constructs containing 2.2 kB of genomic DNA upstream of the coding region are expressed in muscles of the bodywall, pharynx, vulva, uterus, and anus, and also in a few neurons. We have raised antibodies against this protein to analyze its expression. A second CeMyoD-bHLH interacting clone contains a cyclin box (a 32 aa sequence that is shared by all cyclins) and is most similar to Cyclin D (a G1 cyclin involved in the decision to differentiate or re-enter the cell cycle). Progeny of hermaphrodites injected with antisense RNA from this clone have defects in differentiation. We have generated fusion proteins to these two clones to analyze the interaction with CeMyoD by GST pull down and by gel shifts.