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Abstract

The development of an organism is dependent on series of complex sequential events that leads to differential gene expression, resulting in the various cell types necessary to form a full, functional organism. Our lab is interested in the genes involved during organogenesis, specifically in the pharynx of the model organism Caenorhabditis elegans. Composed of only 959 somatic cells, this microscopic nematode serves as a great model to study development. It has therefore been used extensively in studies of conserved genetic pathways. C. elegans develop and reproduce rapidly, are transparent, and have a known cell lineage that allows an individual cell to be traced in its defined patterns of lineage. The M138 strain was chosen to study from a previous mutagenesis screen, which is characterized by extreme deformation of the pharynx resulting in rounding and gaps between muscle cells. We hypothesize this mutation may include the loss of function of cellular adhesion molecules, preventing proper pharyngeal morphogenesis and resulting in L1 larval lethality. To examine possible molecular pathways involved in morphogenesis, the identity of the gene was investigated using complementation tests, genetic, mapping, and RNA interference. Single nucleotide polymorphism mapping revealed the mutation to be located on chromosome I between map units +4 and +8. Complementation analyses with deletion strains further narrowed the chromosomal region to between +4.64 and +9.26 mapping units. RNA interference eliminated likely candidates in the specified region, suggesting the gene has yet to be classified. Future research includes finding the exact sequence of the M138 mutant allele through completion of double-stranded RNA interference, enabling us to perform a transgenic rescue to confirm the identity of the gene.

Introduction

The key to advancement in the scientific world is the ability to “explain the complex visible by some simple invisible,” as stated by the physicist and Nobel laureate Jean Perrin (Carroll, 2006). This concept has been the foundation for the progression of science that has been used by great contributors to the biological realm. Darwin introduced natural selection to explain the diversity of living organisms and how heredity of favorable traits is an essential factor. Watson and Crick’s discovery of DNA then explained how these traits were encoded in the arrangement of just four repeating units called nucleotides. Although these insights described the foundation for the creation of a complex organism, neither natural selection nor DNA can explain how these organisms are made or how they are generated. The field of developmental biology seeks to explain this —how the structure of organisms changes with time.

The molecular foundation of developmental processes has demonstrated a fascinating correlation of Similar mechanisms involved in this progression in all animals. The steps involved in constructing the overall morphology of an organism can be traced back to individual cells, which eventually undergo a series of complex sequential events that lead to their differentiation and ultimate development into a full organism. The fertilization of an egg to form a zygote is just the beginning of a chain reaction of events including regional specification, cell differentiation, morphogenesis, and the growth of the organism (Slack, 2006). 

Our lab focuses on a specific process involved in the development of an organism, called organogenesis, which involves the series of events that lead to the formation of individual organs. The Caenorhabditis elegans pharynx is the organ our lab uses to study development; C. elegans is a hermaphroditic, free-living soil nematode that is used extensively as a model organism for developmental biology questions such as determination of cell fate through differential gene expression, cell signaling, and morphogenesis. Through the use of forward and reverse genetics, we are looking to identify the genes causing mutant phenotypes in the C. elegans pharynx to search for the pathway involved in normal pharynx development.

The Concept of Cell Fate 

In most metazoans, a single diploid cell, known as a zygote, is formed upon the union of a sperm and an egg. It is this one fertilized egg that gives rise to many somatic cells in an embryo through successive rounds of mitosis. Thus, every cell of an organism has the same genome as every other cell; although cells can be structurally and functionally very different. How is it, then, that the sequential cells know what type to become once they mature if they all have the same set of genes? It is differential gene expression from genetically similar nuclei that creates different cell types. The zygote can give rise to hundreds of cell types, such as neurons, blood cells and fat cells. The egg itself has a unique pattern of gene activity that differs from that of the embryonic cells it gives rise to. Genes can either be repressed or expressed in certain cells, resulting in different protein products and therefore different developmental patterns. It is the inherited control of gene activity through epigenetic modifications and microenvironment context that determines whether a certain gene can be switched on in one cell and not in another (Gilbert, 2010). 

Although the fusion of gamete cells stimulates the egg to begin development, there are certain genes that can be expressed without being transcribed by zygotic genes. For example, in C. elegans, only maternally provided gene products are necessary for the initial steps involved in determining cell fate. The asymmetry of early cell divisions, orientation of spindles, and partitioning of macromolecules during the early cleavages are directed by maternal components and are not dependent on zygotic genes (Newman-Smith and Rothman, 1998). The utilization of maternal components is also demonstrated in sea urchin fertilization. Upon sperm entry, there is a rapid increase in protein synthesis without transcription. One mechanism responsible for this rise in the translation of messages contained in the egg cytoplasm is the release of inhibitors from the maternal mRNA (Cormier et al., 2001). For example, the translational repressor 4E-BP inhibits eIF4E from translating mRNA stored in the egg cytoplasm. Upon fertilization, this eIF4E/4E-BP complex is disrupted by phosphorylation and therefore allows eIF4E to regulate the first mitotic divisions of the embryo (Cormier et al., 2001). 

Figure 1: Hermaphrodite and Male C. elegans: (A) Adult hermaphrodite at 100x magnification (bright field) (B) Adult male tail at 400x magnification (bright field)

Cell Differentiation 

Although the embryonic cells may look identical at first, they are able to produce many different cell types through a process called differential gene expression. As will be described, cells create identity by the combinatorial expression and repression of segments of DNA. Differential gene expression results in the presence of specific proteins in each cell type that direct the differentiation of the cell, allowing cells that came from a similar lineage to have different fates. For example, proteins in mature mammalian red blood cells (erythrocytes) cause structural changes that result in the cell losing its nucleus, forming biconcave discs consisting of hemoglobin (Wolpert, 2007). In comparison, neutrophils, a type of white blood cell sharing a common progenitor to erythrocytes, contain certain genes that express different proteins, resulting in changes that lead to a multi-lobed nucleus and a cytoplasm filled with secretory granules (Wolpert, 2007). Despite having different morphology and function, both cells share a similar early lineage.

Multiple factors control the specific mRNAs in the cell at different stages of differentiation, such as the actions of transcription factors, chemical modifications of DNA, and modifications to chromatin proteins that control the pattern of gene expression (Medford et al., 1983). For example, terminal muscle, or myogenic, differentiation requires the coordination of muscle-specific contractile proteins and mRNAs, and there are multiple factors that control these specific mRNAs in the cell at different stages of differentiation (Medford et al., 1983). This is demonstrated by the interactions of the muscle LIM protein (MLP), a crucial component during myogenesis in striated muscle. This protein localizes in different places of the cell during muscle differentiation; it is initially located in the nucleus for the beginning of muscle differentiation and then accumulates in the cytoplasm at later stages. It is the cytoplasmic MLP that associates with actin filaments, thereby regulating the assembly of the myofibril apparatus during muscle maturation (Kong et al., 1997). 

In C. elegans, the protein PHA-4 is an organ identity gene that is involved in the differentiation of cells destined to become the pharynx. Its known transcriptional targets include pha-1 and ceh-22, which regulate genes that are required for pharynx differentiation, as well as myo-2, a structure gene for a pharynx-specific myosin (Newman-Smith and Rothman, 1998). If pha-4 expression is eliminated through mutation or RNA interference, the entire pharynx fails to develop.

Organogenesis 

Organ formation is a vital process in the development of any organism, as this can influence its overall survival. Organs can be thought of as modules, groupings of cells and tissues that serve a particular function for the organism. After the formation of the three germ layers in gastrulation, cells interact to form tissues, which become organized into organs. In order for an organ to develop, intricate cell and tissue interactions need to take place to assure cell growth, differentiation and morphogenesis (Kispert et al., 1998). This process can be divided into two phases. In the first phase of organogenesis, cells are determined to have the fate of becoming a specific organ, while in the second phase, these cells obtain the specific characteristics that are required to make that specific organ functional (Granato et al., 1994). 

For example, during cardiogenesis, when cells are fated to form a heart, many genes are required for development for this organ to effectively circulate blood. The homeobox transcription factor gene Irx4 is required during early embryonic heart development. Irx4 contributes to the establishment of chamber-specific gene expression and is expressed in the ventricle, where it determines the contractile characteristics of the cardiac chamber (Bao et al., 1999). This correct expression of this gene is critical for proper heart function.

Mutations in genes affecting the development of an organ often result in the death of an organism, which further suggests the importance of this process. For example, ventricular hypoplasia occurs due to incomplete or underdeveloped tissues that make up the ventricles of the heart (Park et al., 2010). The transcription factor skNAC is necessary for cardiogenesis since it recruits muscle-specific target genes through physical interactions with DNA-binding partners, and the deletion of skNAC in mice results in ventricular hypoplasia (Park et al., 2010). 

Morphogenesis   

A living organism first begins as many non-living pieces of matter. However, these inanimate bits eventually form something that is living, coming together to construct the tissues of an embryo. Although cells go through the process to become differentiated, they must be distributed in a manner that allows them to create an ordered form. Morphogenesis is defined as the formation of organized animal bodies, and it is through this process that living entities can be established from the non-living (Gilbert, 2010). This process involves precise regulation of cell movement and shape in order to generate the three-dimensional structure of tissues and organs (Portereiko and Mango, 2001). Cell growth, migration, and cell death are all involved in this step (Gilbert, 2010). 

Interactions between various tissues are necessary to ensure that proper functioning is established during development. This can be seen in the formation of tubes, a common component of many organs, that arise from preexisting epithelia into tubular structures or from mesenchymal cells merging to generate tubular epithelia. These events require interactions including the use of signaling pathways, transcription factors, and adhesion molecules (Portereiko and Mango, 2001). Several specialized structures are required in order for these events to occur, but unless they are directed to the right place by morphogenesis, the cells will not be able to survive. For example, in vertebrates, kidney formation is dependent on morphogenetic tissue interactions, such as the signaling molecules between associated mesenchyme and epithelium, which controls organogenesis of three kinds of kidneys (Kuure et al., 2000). It is not only important for these cells to specialize correctly, but that they move into the correct shapes and location in order to form tissues and organs. 

Figure 2: Flow Diagram of Forward and Reverse Genetics: Both the forward and reverse genetic approaches are used to identify the M138 mutation. In forward genetics, the gene is identified based on the amorphous phenotype produced, followed by positional cloning of the mutated gene (phenotype to genotype). Reverse genetics refers to the functional analysis of a gene with known molecular identity (genotype to phenotype). 

One critical component of morphogenesis is cell adhesion, which is necessary for cells to come together and assemble three-dimensional tissues for animals. It is these adhesive systems that are required for individual cells to orient themselves properly though a variety of molecular processes, such as the roles of adhesion proteins as well as cell signaling (Gumbiner, 1996).

Cell Adhesion 

Cell contacts with neighboring cells as well as with the extracellular matrix are important for many developmental processes, having a role in cell migration, morphogenesis, differentiation, proliferation, and apoptosis (Cox and Hardin, 2004). Cells depend on adhesion complexes, such as integrins, which are glycoproteins that form receptors for ligands such as fibronectin, collagen, and laminin. Integrins contain cytoplasmic tails that are responsible for linking the integrins with the actin-containing cytoskeleton, providing a bridge between the two (Kornberg et al., 1992). 

Cell adhesion also has an important effect on cell migration. Varying cells are able to make strong or weak connections based on the quantity and type of adhesion molecules they display, which is important since cells will migrate and localize to places favorable for their adhesion (Ruoslahti and Pierschbacher, 1987). Foty et al., (1996) observed cell sorting and cohesion by spherical aggregates that formed in embryonic chick tissue. The cells that contained the greatest surface cohesion migrated towards the center of the aggregate, since larger cohesiveness requires more energy to force them apart. Their placement in the aggregate was therefore determined based on the strength of cohesiveness the cells possessed (Foty, et al. 1996). 

The maintenance of adult tissue architecture is also largely dependant on the function of cadherins, such as E-cadherin that is expressed in most epithelia (Bailey et al., 1998). The importance of these interactions is demonstrated by their relationship to diseases, such as Barrett’s esophagus, a disorder in which the lining of the esophagus is damaged by stomach acid (Shaheen and Ransohoff, 2002). A decreased expression of E-cadherin was found to be associated in the advancement from Barrett’s esophagus to adenocarcinoma, a lethal cancer in epithelial cells. Failure of cadherins to function correctly allows cell movement and increased proliferation, therefore facilitating tumor progression (Bailey et al., 1998). 

C. elegans as a Model Organism

Model organisms provide insight into understanding processes that are unable to be studied on humans. Human experimentation instigates a variety of problems that stem from violating the ethical principles of using humans as research subjects. It is extremely difficult to design experiments on human beings that will produce the desired scientific information while avoiding ethical contradictions (Rutstein, 1969). For more than a century, the study of non-human experimental systems has been used in various biological disciplines, including genetics and development, molecular and cellular biology, as well as functional genomics and proteomics (Barr, 2003).

Several model organisms have been extensively studied in developmental biology. These include, but are not limited to, C. elegans, Xenopus laevis and Drosophila melanogaster. All possess certain advantages and disadvantages depending on their specific characteristics. For example, while Xenopus laevis (frog) is useful for studying embryonic development, it is difficult to study genetics on this organism due to a slow generation time, of range up to three years (Weinstein and Hemmati-Brivanlou, 1999). 

C. elegans has many characteristics that make it a powerful model system to study several biological processes including apoptosis, cell signaling, cell cycle, cell polarity, gene regulation, metabolism, aging and sex determination (Kaletta and Hengartner, 2006). It has an invariant cell lineage that consists of 959 somatic cells, therefore enabling its complete cell lineage to be determined (Sulston et al., 1983). C. elegans reproduces rapidly and abundantly, developing from an egg to an adult worm in just three days (Kaletta and Hengartner, 2006). It will produce approximately 300 progeny during its lifetime (Hillier et al., 2005). Males can also be found in addition to hermaphrodites, allowing genetic crosses to be done in order to obtain progeny with desired genotypes (Figure 1). Furthermore, this organism’s entire genome has been sequenced, consisting of 19,735 protein-coding genes that are contained between five autosomal chromosomes and one sex chromosome (Hillier et al., 2005). 

Reverse and forward genetics are two approaches that can both be used to study C. elegans. With reverse genetics, the functional analysis of a gene with a known molecular identity is studied, while the forward genetics approach involves the identification of genes based on a mutant phenotype, followed by positional cloning of mutated genes (Barr, 2003). In our lab, the gene causing the amorphous pharynx mutation in the M138 strain is identified using both classical forward genetics as well as reverse genetics (Figure 2).

Pharynx/Morphology of Pharynx 

The pharynx is one of the three major epithelial organs in C. elegans, representing the foregut of the nematode digestive tract (Leung et al., 1999). Food, such as the bacterium E. coli, is pumped in through the buccal cavity by a muscular pharynx, where a specialized cuticle lining the pharynx takes up and grinds the food that is then passed on to the midgut for further digestion (Portereiko and Mango, 2001). Developmentally, the pharynx is derived from the AB and MS somatic founder cells; therefore, the pharynx is polyclonal, meaning it is composed of multiple cell lineages (Mango, 2007). The mature pharynx contains 80 cell nuclei that form five tissues: muscles, epithelial cells, marginal cells, glands and nerves (Granato, 1994); due to cell fusion events, there are fewer than 80 cells. The pharynx can be divided into five distinct regions, from anterior to posterior, the buccal cavity, procorpus, metacorpus, isthmus and terminal bulb (Albertson and Thomson, 1976; Figure 3). 

The pharynx has been shown to be a reliable organ for studying organogenesis. With its complete cell-lineage known, the process of organogenesis can be followed from the earliest steps of development to the terminal steps of differentiation and morphogenesis (Sulston et al., 1983; Mango, 2007). Another advantage of using the pharynx as a model system is the existence of GFP reporters and antibodies that enable individual cell types and developmental stages within the pharynx to be tracked (Mango, 2007). The pharynx will still form as a complete differentiated pharynx even if an embryo cannot undergo normal morphogenesis, therefore making it a valuable tool that allows researchers to focus on the regulation of pharyngeal formation (Mango, 2007). 

Although nematodes do not have a heart or defined circulatory system, previous findings suggest that the nematode pharynx shares functional and molecular similarities to the heart in vertebrates: they are both autorhythmic muscular pumps involved in fluid movement (Mango, 2007; Haun et al., 1998). The similarities to vertebrate heart and pharynx do not end with function. The nkx2-5 transcription factor in invertebrates is orthologous to the transcription factor ceh-22, which is vital for the formation of the pharynx. Interestingly, nkx2-5 was able to efficiently rescue a ceh-22 mutant when expressed in pharyngeal muscle, suggesting an evolutionarily conserved mechanism shared by heart development in vertebrates and the development of the pharynx in nematodes (Haun et al, 1998). 

Early Pharyngeal Specification 

The pharynx is a polyclonal organ, and early cell interactions and specifications are critical in creating a unified organ from two initially dissimilar tissues. After fertilization occurs, the zygote partakes in asymmetric distributions. Different determinants are then partitioned in the daughter cells, which are fated to become either the anterior or posterior part of the pharynx (Hird et al., 1996). After the first asymmetrical division, two cells are produced called AB and P1. AB is the anterior blastomere, and P1  desigantes the posterior blastomere (Priess, 2005). The P1 cell then undergoes an asymmetric division along the anterior/posterior axis to produce the daughter cells EMS and P2, while AB divides transversally to produce the identical ABp and ABa (Priess, 2005; Figure 4). At this point, the four blastomeres that have been formed are the EMS, P2, ABa and ABp, yet only some of the descendent cells of the EMS and ABa blastomeres will be involved in the formation of the pharynx; ABp and P2 contribute to various non-pharynx fates (Mango, 2007). 

The organ identity gene pha-4 is necessary for the formation of both the anterior and posterior sections of the pharynx, affecting pharyngeal cells that derive from both ABa and a daugher of EMS called MS (Mango, 1994). However, different pathways are used by each lineage to activate the crucial gene, pha-4. 

The AB Lineage:  Formation of the Anterior Pharynx

The formation of the anterior pharynx, which is mostly derived from ABa cells, is dependent on two inductive interactions at the 4-cell stage. The first interaction has a mechanism that prevents, while the second interaction induces, pharyngeal development. Both AB descendants, ABa and ABp, contain the Notch-type receptor GLP-1. However, the positioning of the ABa and ABp cells relative to the P2 cell is critical in determining the specification of the anterior pharynx, since GLP-1 is initially only activated in the ABp cell during the 4-cell stage. One major target of Notch signaling in C. elegans is the ref-1 family, which is expressed in response to Notch signaling AB descendants (Neves and Priess, 2005; Figure 4). While the ABp cell gives rise to ectodermal cells, it is the ABa cell that is fated to contribute to anterior pharyngeal cells (Gilbert, 2010; Priess, 2005). 

First, the inhibiting GLP-1/Notch interaction in the ABp cell will be described, in which the formation of the anterior pharynx is suppressed. The posterior location of the ABp cell allows it to be in contact with the P2 cell containing APX-1, a delta-like ligand, which binds the GLP-1/Notch on ABp. This first interaction is necessary in suppressing pharynx formation in this cell by repressing tbx-37 and tbx-38, which is mediated by the ref-1 family (Neves and Priess, 2005; Figure 4). These two genes encode for transcription factors that are essential for AB descendants to produce pharyngeal tissue by activating pha-4, the organ identity gene. Therefore, by repressing tbx-37/38, the competence of the ABp cell to produce pharyngeal tissue is restricted, and instead gives rise to neurons and hypodermal cells  (Good et al., 2004; Gilbert, 2010; Figure 4). 

The second Notch-signaling interaction occurs at the 12-cell stage, where the MS blastomere (descendant of the EMS cell) touches the two ABa granddaughters. While the first signal prevented pharyngeal specification by the repression of tbx-37 and tbx-38, this second signal acts in contrast to produce pharyngeal tissue. The ABa descendants still contain a GLP-1/Notch receptor, which promotes the expression of lag-1, which in turn activates the ref-1 family of transcription factors (Smith and Mango, 2007). These then activate the organ identity gene pha-4, which leads to the formation of the anterior pharynx (Smith and Mango, 2007; Figure 4). In addition to activation by LAG-1, pha-4 is also activated by tbx-37/38 in the ABa cells. Although REF-1 represses the expression of tbx-37/38 in the ABp descendants, this repression does not occur in the ABa cells. The reasoning for this is due to the timing in which ref-1 is expressed (Neves and Priess, 2005). During the second

Figure 3: The C. elegans Pharynx: Anatomical sections of the pharynx from anterior to posterior the buccal cavity (orange), procorpus (blue), metacarpus (green), isthmus (red) and terminal bulb (yellow). 

Figure 4: Cell Signaling Pathways in Pharyngeal Development: Genes known to be involved in the activation of organ identity gene pha-4 leading to commitment to anterior (green) or posterior (pink) pharynx fate. Lines indicate cell divisions. Genes colored in peach are more specific to anterior formation while purple specifies posterior pharyngeal formation. Genes colored in yellow have roles in both anterior and posterior development. Adapted from Sadozai (2011) and Charron (2010) using Priess (2005), Neves and Priess (2005), Good et al. (2004), Gilbert (2010), Smith and Mango (2007), Bowerman et al. (1992), Broitman-Maduro et al. (2006), Lin et al. (1995).

interaction, ref-1 is expressed too late to prevent tbx-37/38 expression in ABa descendants at the 24-cell stage, allowing the expression of genes necessary for pharynx formation. While tbx-37 and tbx-38 are expressed at the 24-cell stage, the expression of ref-1 does not occur until later during the 26-cell stage (Neves and Priess, 2005; Figure 4).  In essence, these reciprocal interactions ensure that the correct number of ABa cells become part of the pharynx.

EMS Lineage: Formation of the Posterior Pharynx

While the AB lineage requires glp-1 Notch signaling, the EMS lineage is instead dependent on the signaling from the maternal genes skn-1 and pop-1 in order to produce posterior pharyngeal cells  (Bowerman et al, 1992; Figure 4). 

In contrast to the AB, the division of P1 is asymmetric and is responsible for the differences in fates of the EMS and P2 blastomeres (Rose and Kemphues, 1998). SKN-1 is required to specify EMS fate. In skn-1 mutants, the muscle, pharynx, and intestine that are normally produced by MS and E are absent (Rose and Kemphues, 1998). Instead, the EMS descendants acquire a C fate, which produces muscle tissue, hypodermis and neurons in the place of pharynx cells (Rose and Kemphues, 1998; Bowerman et al., 1992). To promote further specification in MS, SKN-1 activates transcription of the GATA factors med-1,2 in EMS (Broitman-Maduro et al., 2006). The target gene of MED-1,2 is tbx-35, which encodes a member of the T-box class of transcriptional regulators that specify MS fate, including the activation of the organ identity gene pha-4  (Broitman-Maduro et al., 2006; Figure 4). 

The maternal gene pop-1 is responsible for cell fate decisions after the eight-cell stage in the EMS blastomere. POP-1 is more abundant in anterior cells compared with their posterior sisters, having a role in specifying anterior identity (Labouesse and Mango, 1999). The anterior/posterior division of the EMS cell results in MS being located more anteriorly, resulting in a higher level of POP-1 protein (Lin et al., 1998). POP-1 is thus down regulated in posterior daughter cells by LIT-1, which is required in distinguishing sister-cell fates (Bowerman et al, 1992). POP-1, which blocks the activation of tbx-35 in a Wnt/MAPK-dependent manner, is repressed in E (Broitman-Maduro, 2006; Figure 4). Therefore, the activity of pop-1 is necessary for the EMS daughter cells to acquire different fates. The MS descendent produces mesodermal tissue, including pharyngeal cells that make up the posterior pharynx due to reduced activity of pop-1, while the E descendant acquires an endodermal fate in its presence  (Sulston et al.,1983; Lin et al., 1995).

In addition to the formation of the posterior pharynx, the MS blastomere also has a role in signaling to the ABa descendants of the previously described AB lineage, in which the anterior pharynx is formed.

Pharyngeal Morphogenesis

Morphogenesis is a critical step in establishing the formation of the pharynx. The formation of the pharynx involves organizing eight sets of cells that are joined end to end by adherens junctions (Albertson and Thompson, 1976). Pharyngeal morphogenesis can be divided into three stages: reorientation of anterior pharyngeal cells, formation of an epithelium by the buccal cavity cells, and an anterior and posterior movement of the pharynx and epidermis, respectively (Portereiko and Mango, 2001; Figure 5). 

Figure 5: Pharyngeal Morphogenesis. (A) Pharynx cells (green) form a cyst and are separated from arcade cells (yellow). (B) Anterior pharynx cells undergo “reorientation,” changing their polarity to align with arcade cells. (C) “Epithelialization” results in pharynx and arcade cells forming continuous epithelium. (D) “Contraction” stage pulls cells of pharynx, buccal cavity, and epidermis anteriorly (from Mango, 2007).

Prior to pharyngeal extension, the pharyngeal primordium forms a cavity deep within the embryo in which it is separated from arcade cells by a basement membrane (Figure 5A). During Stage I of pharyngeal extension, known as “reorientation,” the anterior pharyngeal cells alter their morphology from a cyst to a short tube by reorienting their apicobasal polarity (Figure 5B). This movement is important for the formation of a continuous epithelium in Stage II since it aligns the pharyngeal epithelial cells with the arcade cells (Figure 5B, C). Additionally, cells located at the anterior tip of the pharyngeal primordium lose cell contacts with some of their neighbors. This adjustment is necessary in removing the physical barrier that these cells inflict between the developing pharyngeal lumen and the buccal cavity (Portereiko and Mango, 2001). 

The second stage of pharyngeal extension is “epithelialization.” The buccal cavity forms adheren junctions that are responsible for connecting the buccal cavity to the pharynx and epidermis. This epithelialization of the arcade cells produces a continuous epithelium (Portereiko and Mango, 2001; Figure 5C).

At the third stage, “contraction,” the cells of the pharynx, buccal cavity, and epidermis are already connected. They are then pulled together by a local contraction, which causes the pharynx to move forward while simultaneously resulting in backward movement of the epidermis (Figure 5D). It is suggested that arcade cells are responsible for the occurrence of this contraction. This was demonstrated when laser ablation of arcade cells resulted in the absence of a contraction phase, implying their importance for this process (Portereiko and Mango, 2001).

Cell Adhesion in C. elegans and Its Pharynx

C. elegans has several adhesion complexes that are related to those in vertebrates, which makes this organism a great model for studying the regulation and function of adhesive structures in vivo. Included in these adhesion complexes are epithelial apical junctions, dense bodies, fibrous organelles, and a glycoprotein complex (Cox and Hardin, 2004). Cell adhesion has a critical role in the pharyngeal morphogenesis in C. elegans. In order for the cells to change shape or be rearranged, signaling processes must be sent from within the cell to adhesive junctions located at the plasma membrane, where the forces are transmitted to neighboring cells or the extracellular matrix (Gumbiner, 1996). 

Worm epithelial cells contain two adhesion complexes, the cadherin-catenin (CCC) and the DLG-1/AJM-1 (DAC) complexes. Together, these are known as CeAJ (C. elegans Apical Junction) (Labouesse, 2006). The apical domain of the CeAJ is formed by 

A cadherin complex, composed of the HMP-1, HMP-2, and HMR-1 proteins. These are members of the α-catenin, β-catenin, and classical cadherin families, respectively, which localize to adherens junctions in C. elegans embryos.

This cadherin complex functions to anchor the actin filament bundles to the adherens junctions in hypodermal cells, translating the force of bundle contraction into cell shape change (Costa et al., 1998). In embryos lacking hmp-1 and hmp-2, the mutants displayed abnormally bulged dorsal surfaces, hence being named after a “hunchback” phenotype. However, in hmr-1 mutants, the hypodermis failed to fully enclose the embryo, displaying a “hammerhead” phenotype (Costa et al., 1998). 

The apical domain remains distinct from the basal domain in that the latter is characterized by the presence of the DLG-1 and AJM-1 proteins. DLG-1 regulates the localization of AJM-1 to a distinct apical domain that is basal to the HMR-HMP cadherin complex. This localization occurs independently of the HMR-HMP complex. Each complex is able to localize normally in the absence of the other. Although the precise mechanism of the ajm-1 gene is unknown, it is speculated that this basal domain serves as a permeability barrier for maintaining the paracellular seal, similar to the permeability barrier, the septate junction, in Drosophila (Koppen et al., 2001).

Gap in Knowledge

Despite all the information that has already been obtained on the developmental processes in C. elegans, there are still extensive gaps in knowledge regarding possible genes involved in pharyngeal morphogenesis. Our lab has produced more than 200 defective pharynx phenotypes in the C. elegans pharynx using ethylmethanesulfonate (EMS) to gain more insight on the abnormal pharynx muscle morphology for these worms. The specific strain that was studied in this thesis is the M138 strain, which exhibits an extreme deformation of the pharynx causing larval lethality. The goal of our lab is to identify the exact location and identity of the alleles responsible for this mutant phenotype. Previously, our lab had narrowed down the location of the allele to be on chromosome I, between the regions of +1 and +8. We hypothesize that through genetic mapping and complementation analysis we will narrow down the location of the gene to within a few map units. Then, using RNA interference (RNAi), the location will be identified and then confirmed using further complementation, in which the gene will be sent for sequencing.