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Abstract

Embryogenesis is a fascinating process during which a single cell transforms into a multi-cellular hybrid of organs and tissues. Our lab investigates the underlying genetic framework that drives organogenesis through our study of the model organism, Caenorhabditis elegans (C. elegans). C. elegans is a free-living nematode that has a pronounced pharynx, which is ideal for studying organogenesis from the incipience to the end of differentiation and morphogenesis. We canspecifically locate the mutation responsible for producing a short pharynx phenotype observed in the mutant line of C. elegans called M77, which is larval lethal. We described the mutant pharyngeal phenotype through light microscopy, immunocytochemisty, and we utilized complementation tests and genetic mapping to identify the location of the mutant gene, each procedure proving some explanation for a possible mechanistic pathway for morphogenesis. We also aimed to genetically balance the M77 strain. We were able to narrow down the location of the mutant gene to chromosome III between -3.10 and -4.47 mu that effectively puts the mor-1 gene, which was previously thought to be the possible gene causing the M77 phenotype, outside this mu range. However, we were not able to find any gene that exhibits phenotypes similar to the M77 mutation gene within the range of -3.10 and -4.47 mu. We believe that it might be a gene that has not been previously described and therefore we might be the first ones to describe this gene. We also successfully balanced the M77 strain. In addition, we observed that a 7% ethanol treatment suppressed larval death and the mutant worms progressed even through the L1 developmental stage. Our future goals include determining the identity of the M77 mutant gene through further complementation analyses, interval mapping, sequencing, and conducting a confirmation of the mutant gene identity through a transgenic rescue of the mutant worms. Moreover, we hope to determine the molecular pathway through which the M77 gene functions.

Introduction
Developmental biology is the study of processes and natural phenomena that occur during an organism’s growth and differentiation. It is primarily concerned with the period of “becoming” rather than the period of “being” (Gilbert, 2006). More specifically, developmental biology seeks to understand the fascinating transformation of a single cell into a multi-cellular organism composed of tissues and organs. Generically, in virtually all multi-cellular organisms, the process of development begins with the fusion of an egg with a sperm, a process called fertilization. Fertilization occurs to yield a diploid zygote that matures to subdivide, specialize, and produce a multi-cellular organism such as a Homo sapiens (Gilbert, 2006; Ferrier, 2009; Charron, 2010).
Developmental biology research is dedicated to understanding the causes behind numerous congenital diseases, such as Holt-Oram, which is an abnormality of the upper limbs and heart (Fan et al., 2003; Mori & Bruneau, 2004). Moreover, it has been key in shaping our current understanding of cancer. Developmental biologists are taking steps to identify signaling pathways that control tissue growth and organization (Edwards, 1999). Developmental research has elucidated the vital importance of the period known as organogenesis for the proper development of an organism. Moreover, it has highlighted the potential risks of certain irregularities that may occur during the period of embryogenesis. These risks may result in major birth disorders, such as Holt-Oram syndrome (Fan, Liu, & Wang, 2003; Mori & Bruneau, 2004).
 It is the focus of this senior thesis project to understand the genetic mechanisms and regulatory machinery governing and conducting the transformation of a single cell into a functional multi-cellular organ. The purpose of this research is to expand the findings of the previous research conducted by Andrew Ferrier by using the fore-gut (pharynx) of the microscopic nematode, Caenorhabditis elegans (C. elegans), as a model to further genetically map and specifically locate the mutation responsible for producing the short pharynx phenotype observed in the mutant line of C. elegans called M77 with enough precision to identify its chromosomal location. The pharynx is not unique to C. elegans. It is found in humans as well; the pharynx is part of the vertebrate alimentary canal and it extends to the larynx (Daniels, 2007; Tortora & Nielsen, 2009). C. elegans have an elongated, cylindrical pharynx that has a terminal bulb at one end. This research seeks to determine the genetic and molecular cause of the short pharynx phenotype resulting from a mutation in M77 worms. Simultaneously, we are aiming to preserve the mutated strain by genetically balancing the M77 allele with a deletion strain.
C. elegans As A Model Organism
Utilization of a model organism in biological research to study and understand a particular phenomenon and then, apply the learned knowledge to other organisms is vast and immensely common.  The use of model organisms is largely possible because the metabolic and developmental mechanisms and pathways that exist today have largely evolved from a common point (Barr, 2003; Kaletta & Hengartner, 2006).  Therefore, various organisms can be studied to better understand pathways that have been conserved over time.  The usage of a model organism in biological research makes it easier for scientists to conduct research to explore and access the root causes of certain human diseases, which otherwise would require human experimentation (Barr, 2003; Kaletta & Hengartner, 2006).  Human experimentation is not a viable research option because it requires the knowing consent of the subjects and depending on the research topic, the use of human subjects might be unethical.  Moreover, because embryonic development occurs in the uterus, human experimentation limits the methods that researchers can use to investigate a topic (Rutstein, 1969).  
In developmental biology, there are several model organisms that have been extensively used and studied.  These studies have provided us with valuable information about the genetic and molecular pathways coordinating development.  Already developed model organisms include chicks, sea urchins, Drosophila melanogaster, mice, Xenopus laevis, zebrafish, and C. elegans (Barr, 2003; Gilbert, 2006; Kaletta & Hengartner, 2006).  In our study, we used C. elegans as our model organism for various reasons.
There are ample advantages in using any of the model organism listed above.  However, the use of model organisms such as sea urchins and Drosophila pose certain disadvantages (Gilbert, 2006).  Sea urchins are quite difficult to cultivate and manage in laboratory conditions beyond a certain stage.  Drosophila undergoes a complicated developmental process, which makes the research problem more difficult to assess and solve (Gilbert, 2006).  In fact, the complicity of Drosophila’s developmental process drove the Nobel-Prize winner Sydney Brenner and his colleague to spearhead another search in the quest of finding a simple model organism (Gilbert, 2006).  C. elegans is a simple model that overcomes the disadvantages of aforementioned model organisms (Gilbert, 2006).  It enables the researchers to identify each gene in the C. elegans’ genome and trace the lineage of each C. elegans’ cell if desired.
C. elegans are about a millimeter long (Brenner, 1974; Gilbert, 2006).  They are small, free-living, non-parasitic, soil nematodes that were introduced as a model organism in 1974 by Sydney Brenner, “The genetics of Caenorhabditis elegans,” to study neurobiology and developmental biology.  C. elegans is one of the simplest multicellular eukaryote that has recently gained popularity in the scientific community.  It is being used to study various insightful biological processes, such as apoptosis, cell signaling, cell cycle, cell polarity, gene regulation, metabolism, aging and sex determination (Kaletta & Hengartner, 2006).  In fact, programmed cell death, which is an evolutionarily conserved process used by multicellular organisms to eliminate unwanted cells, has been extensively studied in C. elegans (Adams & Cory, 1998; Conradt & Horvitz, 1998; Jacobson, Weil, & Raff, 1997; Metzstein, Stanfield, & Horvitz, 1998; Spector, Desnoyers, Hoeppner, & Hengartner, 1997).  According to Spector and his colleagues, mammalian Bcl-2 family members might control apoptosis in an analogous way as CED- 9 in C. elegans (Spector et al., 1997).
Similarly, many other studies have revealed a remarkable biological similarity between C. elegans and other mammals.  In fact, humans share numerous biological properties with C. elegans that have conserved and preserved various intact mammalian biological processes, such as program cell death or apoptosis (Metzstein et al., 1998).  Consequently, scientists have taken advantage of this fact and conducted various research studies on C. elegans.  The studies have led to innumerable, crucial discoveries not only in the field of biology but also in the medical field.  For example, the first presenilin discovery was made through a research study on C. elegans in 1993.  Presenilin has been identified as a component of γ-secretase complex, which is a key Alzheimer’s disease’s target (De Strooper et al., 1999; Kaletta & Hengartner, 2006).  In fact, mutated human presenilin genes lead to the most frequent and aggressive forms of Alzheimer’s disease (Wittenburg et al., 2000).  C. elegans is used as a model organism to study the genes regulating the developmental process. 
                                   
Figure 1:  Life cycle of C. elegans at 22ºC (room temperature): Constructed using information from WormAtlas. C. elegans life cycle is temperature-dependent and they usually live about 2-3 weeks at room temperature. C. elegans goes through four larval stages before becoming a reproductive adult. However, in the absence of food or crowdedness, C. elegans can enter the dauer larval stage, which allows them to survive up until 4 months.

C. elegans has numerous attractive features that make it a powerful model organism for genetic studies.  Firstly, it is very easy to cultivate (Kaletta & Hengartner, 2006).  Even though in its natural environment it feeds on various bacteria, it can easily be grown and maintained in laboratory conditions with a simple diet of Escherichia coli (Barr, 2003; Brenner, 1974; Kaletta & Hengartner, 2006).  We used the OP50 strain of E. coli in our study.  Secondly, C. elegans are transparent.  They are easy to study under the microscope (Kaletta & Hengartner, 2006; Sulston, Schierenberg, White, & Thomson, 1983).  In fact, each cell can be individually observed without needing to cut and fix it to a slide.  This property is known as single cell resolution, which means you can look at the animal under a microscope and see every cell within it without having to do anything special or extraneous to the animal.  Moreover, the research can clearly trace the entire cell lineage through C. elegans’ transparent body (Sulston et al., 1983).  Thirdly, C. elegans possesses a fixed number of cells that are invariant, which essentially means that they are nearly identical to each other (Jorgensen & Mango, 2002; Labouesse & Mango, 1999; Sulston et al., 1983).  This property is quite advantageous for carrying out experiments because there is very little room for randomness (Sulston et al., 1983).  Fourthly, C. elegans has a very short life cycle of about three days at room temperature and it reproduces very rapidly giving birth to more than 300 progeny (Barr, 2003; Kaletta & Hengartner, 2006).  Thus, scientists are able to rapidly conduct experiments which otherwise would take time if C. elegans took a longer period to grow and reproduce (Figure 1).  Fifthly, C. elegans’ small size makes it very easy to handle in the laboratory.  It is small enough to be able to be cultivated in the amount of hundreds of worms on a single plate (Kaletta & Hengartner, 2006).  It diminutive size also enables it to be easily transferred into a microfuge tube.  However, C. elegans are big enough to be individually picked using a standard stereo microscope and platinum wire.
Furthermore, C. elegans have a very interesting gender division.  C. eleganshave two sexes: hermaphrodites with 959 somatic cells and males with 1031 somatic cells (Gilbert, 2006; Kaletta & Hengartner, 2006; Schedin, Hunter, & Wood, 1991; Sulston et al., 1983).  Males are important for genetic studies because they allow us to introduce different alleles into a population by inducing the worms to sexually mate with a worm carrying the desired allele (Figure 2).  C. elegans are easy to preserve by freezing desirable strains in liquid nitrogen (Kaletta & Hengartner, 2006).  Finally, C. elegans’ genome has been completely sequenced (Barr, 2003; Hillier, Coulson, Murray, Bao, & Sulston, J. and R. H. Waterston, 2005; Rose & Kemphues, 1998; Sulston et al., 1983) .  Therefore, we have a vast amount of information for apprehending the molecular organization of an organism.  Its genome is packed into six chromosomes consisting of about 19,000 genes out of which about 40% have been identified to be homologous to other organisms, including humans (Genome sequence of the nematode C. elegans: A platform for investigating biology.1998; Cooper & Hausman, 2006).  Essentially, these characteristics make C. elegans a powerful model organism for developmental genetic studies. 
The Importance of Understanding Pharyngeal Development
As mentioned before, C. elegans’ pharynx is the foregut, which is an essential part of the digestive tract of the worm composed of muscular epithelial tissue (Albertson & Mango, 2007) . It is a narrow, tube-shaped organ, which is Thomson, 1976; Horner et al., 1998). Since the publication of ultra-structural studies by Alberston and Thomson (1976),
a great amount of information was elucidated about the C. elegans’ pharyngeal anatomy and structure (Mango, 2007).
  

Figure 2: (A) Adult hermaphrodite and (B) adult male with an arrowhead tail at 100x magnification (bright field): (A) Adult hermaphrodite and (B) adult male with an arrowhead tail at 100x magnification (bright field).

The pharynx is a neuromuscular organ that functions as a rhythmic pump to grind and consume bacteria (Horner et al., 1998; Mango, 2007). Food is digested in the pharynx and then, the food is further passed down the gut (Albertson & Thomson, 1976). C. elegans’ pharynx has a two-lobed, linear structure. It is divided into different sections from the anterior to the posterior end (Mango, 2007). For example, it is composed of the buccal cavity, procorpus, metacorpus, isthmus and terminal bulb (Figure 2) (Mango, 2007). Moreover, a basement membrane separates the pharynx and demarcates it from other C. elegans’ tissues (Albertson & Thomson, 1976). In fact, the basement membrane marks out C. elegans’ pharyngeal nervous system as an entirely separate entity that consists of five different types of motor neurons and six different types of inter-neurons (Albertson & Thomson, 1976). C. elegans’ pharynx’s composition, like the makeup of the foreguts of other complex organisms such as humans, consists of many distinct cell types. It is polyclonal, which means that the pharynx is composed of multiple cell types (Mango, 2007).  These cell types include epithelial cells (9), gland cells (5), marginal cells (9), muscle cells (34), and neurons (20) (Albertson & Thomson, 1976).
C. elegans’ foregut (pharynx) has become a great tool to study organogenesis that occurs during the developmental stage due to various, multiple appealing features of the pharynx (Mango, 2007). For example, organogenesis can be observed in the pharynx from the beginning to the end (Mango, 2007). Scientists are able to even trace the last steps of differentiation and morphogenesis because C. elegans are transparent and their complete cell lineage is known (Mango, 2007). In fact, the structural anatomy of the pharynx has been well studied and keenly characterized (Albertson & Thomson, 1976). Using certain antibodies and green fluorescent protein (GFP), researchers can mark and track individual pharyngeal cell types and identify various developmentalstages the pharynx undergoes during its development (Mango, 2007). In addition, C. elegans’ ability to develop a normal, differentiated pharynx even in the face of complications, such as abnormal morphogenesis, in the developmental process allows scientists to focus on components that directly modulate pharyngeal formation (Mango, 2007).


Figure 3: Anatomical sections of C. elegans pharynx: C. elegans pharynx up and its Schematic drawing lower illustrates anatomical sections of pharynx from anterior to posterior: Buccal cavity (lower yellow); Procorpus (lower green); Metacorpus (lower red); Isthmus (lower blue); Terminal bulb (lower orange).
 
Scientists can disregard the possibility of indirectly produced pharyngeal abnormalities by C. elegans’ intracellular machinery that regulates other developmental processes. Finally, there are several mechanistic characteristics and evolutionary pathways that C. elegans’ pharynx utilizes that are commonly applied by higher organisms, such as humans (Mango, 2007). Therefore, C. elegans’ pharyngeal development during organogenesis is ridden by similar developmental obstacles as other higher organisms.
C. elegans’ pharynx has shown to have mammalian orthologues or Orthologs, which are genes in different organisms that originate from a single gene in those organisms’ last common ancestor. The importance of these genes is that they often maintained identical biological roles and functions in the organisms of modern day (Remm, Storm, & Sonnhammer, 2001). For example, it is thought to be analogous to not only the esophagus and stomach (foregut) of other organisms but it is also found to be analogous to the human heart (Mango, 2007). Both the pharynx and human heart are shaped like a muscular tube that moves essential materials through the body through rhythmic contractions needed to sustain life. There are some key differences between the human heart and C. elegans’ pharynx. While the heart pumps blood throughout the body carrying vital nutrients and minerals, the pharynx grinds and ingests bacteria and then, passes the food further down the gut of the worm. However, both organs rely on electrical impulses to maintain synchronous muscle contractions. They do not require any input from the nervous system in order to carry out their function (Haun, Alexander, Stainier, & Okkema, 1998; Mango, 2007). Moreover, they operate using similar types of potassium-and voltage-gated calcium channels (Mango, 2007).
In addition, there have been orthologous transcriptional factors that have been identified in both the human heart and C. elegans pharynx. These transcriptional factors are thought to be essential for the normal development of each of the organ. For example, both the heart and pharynx utilize transcriptional factors belonging to the NKX transcriptional factor family in order to develop (Mango, 2007; Okkema, Ha, Haun, Chen, & Fire, 1997). The NKX transcriptional factor family is a phylogenetically conserved group of homeobox genes, which are approximately 60 amino acid sequences and encode transcriptional regulatory proteins that have vital roles during developmental stages of an organism (Lints, Parsons, Hartley, Lyons, and Harveyand, 1993; Slack, 2006). It has been found that a gene called the ceh-22 gene encodes for an NK-2 class homeodomain protein in C. elegans (Okkema & Fire, 1994). NK-2 class homeodomain protein is required for normal pharyngeal development. Moreover, the ceh-22 gene is functionally similar to Drosophila’s tinman and vertebrate’s Nkx2.5, which are involved in the cardiac muscle formation in Drosophila and vertebrates (such as zebrafish and humans) respectively (Okkema et al., 1997). Therefore, the ceh-22 gene is responsible for muscle development in C. elegans (Okkema, Ha, Haun, Chen, & Fire, 1997). Moreover, Haun (1998) and his colleagues have successfully shown that worms with a mutation in the ceh-22 gene (the ceh-22 mutants) can be effectively rescued through the expression of the vertebrate’s Nkx2.5, which was introduced in the mutant worms, in pharyngeal muscle.

 
Figure 4: A potential pathway for the Ceh-22 and Nkx2.5 gene: The ceh-22 gene and Nkx2.5 share a similar role in C. elegans and vertebrates respectively. Nkx2.5 can activate the ceh-22 gene and the pharyngeal muscle protein, myo-2, to form pharynx.

The effective rescue of the ceh-22 mutants suggests that the ceh-22 gene and the pharyngeal muscle protein (myo-2) are both activated by the vertebrate’s Nkx2.5 (Haun et al., 1998). Refer to Figure 4 for a hypothesized working pathway for the ceh-22 gene and Nkx2.5. Consequently, these findings suggest that the regulating pathways involved in pharyngeal development in C. elegans share common features with the pathway that regulates the cardiac muscle formation in higher organisms, such as zebrafish and humans.
There are other aspects of C. elegans’ pharyngeal development that are linked to the development of other organs in other higher organisms. For example, pha-4 gene, whose expression is essential for the pharyngeal development, has been found to be an orthologue to the FORKHEAD gene family and the FoxA gene in Drosophila melanogaster and mammals respectively (Carlsson & Mahlapuu, 2002) . The FORKHEAD gene family and the FoxA gene are crucial for gut development (Gaudet & Mango, 2002; Kalb et al., 2002). Furthermore, another orthologue of the pha-4 gene is the FoxA2 transcriptional factor, which has also been found to play a crucial role in gut formation in all organisms that have been studied up-to-date (Carlsson & Mahlapuu, 2002). In addition, the pha-4 gene also encodes an HNF-3 homolog Ce-fkh-1, which is expressed in all the muscles of the pharyngeal precursor and it has been determined to regulate their fate. Moreover, the HNF-3 homolog ce-fkh-1 has been discovered to be involved in gut development in other organisms (Horner et al., 1998). These results discovered by various researchers collectively illustrate that the transcription factors have been conserved between organisms. Therefore, the underlying genetic mechanistic pathway governing pharyngeal development must be also evolutionarily preserved in C. elegans.
C. elegans pharyngeal development is not only suggested to be similar to the development of the heart in vertebrates but it has been claimed to be similar to kidney tubulogenesis. For example, both the pharynx and kidney undergo apical to basal polarity rearrangement in tubulogenesis (Portereiko & Mango, 2001). These results support the continued efforts to understand pharyngeal development in C. elegans in order to gain insight into the development of complex organs in other higher organisms, such as ourselves.

The Formation of the Pharynx
Aforementioned, the pharynx of C. elegans is formed poly-clonally, which means that multiple progenitor cell types are involved in the formation of the pharynx. Early developmental patterning starts with the sperm fertilizing the egg to form a zygote (the P0 cell) (Rose & Kemphues, 1998). The site of penetration of the oocyte by a sperm becomes the anterior end of the zygote, while the opposite side becomes the posterior end of the zygote (Goldstein & Hird, 1996). The zygote undergoes an asymmetrical division, which results into an anterior blastomere (AB) and a posterior blastomere (P1) (Priess, 2005). While the AB cell divides into ABa and Abp blastomeres, the P1 cell divides into EMS and P2 blastomeres (Gilbert, 2006; Mango, 2007; Priess, 2005). At this stage, there are four types of blastomeres. However, only two of the blastomeres, the ABa and EMS blastomeres, eventually divide to make the pharynx (Mango, 2007). The other two blastomeres, ABp and P2 blastomeres, do not partake in the further development of the pharynx (Mango, 2007). The ABa blastomere produces the anterior pharynx cells whereas the EMS cell produces the posterior pharynx cells. However, the two blastomeres give rise to pharyngeal cells through a completely different molecular pathway. ABa relies on intracellular communication between cells and glp-1 RNA (a Notch receptor orthologue and maternally contributed gene) to produce components that eventually give rise to the anterior pharyngeal cells (Mango, 2007; Priess, 2005). On the other hand, the EMS utilizes the maternally contributed genes skn-1 and pop-1 to produce the components that eventually give rise to the posterior pharyngeal cells (Bowerman, Eaton, & Priess, 1992; Lin, Thompson, & Priess, 1995; Mango, 2007). In addition, it is worth noting that both the ABa and EMS cells also contribute to formation of non-pharyngeal cells, such as the body wall muscle, epidermis, gonad, intestinal cells, and neurons (Sulston et al., 1983).

The Formation of the Anterior Pharynx
C. elegans’ pharynx originates from the two descendents of AB and P1, namely ABa and EMS respectively (Mango, 2007).  From the point of development of ABa, ABp, P2 and EMS (which henceforth will be referred to as the 4-cell stage), ABa and EMS blastomeres essentially begin to control the development of both pharyngeal cells as well as non-pharyngeal cells, such as epidermis and neurons.  At the 4-cell stage, the fate of the divisional remainders of AB, ABa and Abp, are not yet determined.  Therefore, ABa and ABp can act like pluripotent stem cells and they can differentiate into any worm body-cell, such as cells that make up the epidermis and body wall muscle (Sulston et al., 1983).  However, ABa and ABp, express GLP-1 or LIN-12 receptors, which are Notch receptors orthologues.  Notch signaling mechanisms and pathways have been evolutionarily conserved as intact processes during the development of various organisms and Notch signaling has been observed to play a vital role in determining cell fate as well (Artavanis-Tsakonas, Rand, & Lake, 1999).  Likewise in pharyngeal development, Notch signaling pathway determines the anterior pharyngeal cell fate at the 4-cell stage.  According to Good et al. (2004), the divisional remainders of AB initially adopt the fate to turn into an ectodermal cell.  The posterior daughter of AB, ABp, expresses the glp-1/Notch receptor.  It interacts with P2 via Notch signaling pathway.  ABp expresses a glp-1/Notch ligand, which is encoded by a maternally inherited gene called apx-1.  Through Notch signaling ABp is confined to a fate to become into ectodermal cells (Good et al., 2004; Mango, Thorpe, Martin, Chamberlain, & Bowerman, 1994).  The glp-1 targets the ref-1 family of transcriptional factors, which when activated inhibit the activity of a pair of redundant T-box genes, tbx-37 and tbx-38 transcriptional factors.  Tbx-37 and tbx-38 transcriptional factors are essential for the pharyngeal development because they activate the pha-4 gene, which determine pharyngeal cell identity (Good et al., 2004).  To recap, the glp-1/Notch receptor activation in ABp via Notch signaling pathway activates ref-1 family of transcriptional factors, which in turn suppresses the tbx-37 and tbx-38 activities, which monitor the expression of the pha-4 organ-identity-determining gene. 
On the other hand, the anterior daughter of AB, ABa, does not come into contact with P2 and therefore, it does not become confined to an ectodermal cell fate.  It gains an ectodermal cell fate during the 12 to15-cell stage.  During the 12 to 15-cell stage, the granddaughters of ABa interact with a descendent of EMS, MS, via Notch signaling pathway.  The interaction between MS and ABa descendants activates Lag-1, which in turn induces the expression of ref-1 family of transcription factors, which activates the pha-4 gene (Smith & Mango, 2007).  The activation of pha-4 gene leads to the formation of anterior pharynx (Smith & Mango, 2007).  In this case, the pha-4 gene expression is also induced by tbx-37 and tbx-38 transcriptional factors along with its activation by lag-1.  The lag-1 activation induces the expression of ref-1 family of transcriptional factors, which as previously mentioned actually inhibits the expression of tbx-37 and tbx-38 transcriptional factors.  The inhibition of tbx-37 and tbx-38 transcriptional factors leads to the deactivation of the pha-4 gene (Smith & Mango, 2007).  Therefore, the activation of ref-1 family of transcriptional factors has a negative influence on the activation of the pha-4 gene.  However, this is not observed in this scenario oddly enough.  The reason for this anomaly is that the activation of various components of this mechanism occurs at different times.   The tbx-37 and tbx-38 transcriptional factors are expressed during the 24-cell stage while the expression of ref-1 family of transcriptional factors occurs later during the 26-cell stage (Neves & Priess, 2005).  Therefore, ref-1 does not inhibit the expression of tbx-37 and tbx-38 during the 24-cell stage.  Hence, the pha-4 gene expression is activated due to the expression of tbx-37 and tbx-38 and activation of lag-1.  It is clear that the anterior pharyngeal formation is a complex process that entails intricate time-specific gene interactions and spatially induced Notch signaling.  
In addition to playing a crucial role in the anterior pharyngeal cell specification, the MS, produced during the 7-cell stage, is also a mesodermal precursor and it is involved in the posterior pharyngeal cell formation.  In general, the MS blastomere is responsible for the development of mesodermal cell types including pharyngeal cells, body muscles and coelomocytes (Broitman-Maduro et al., 2009; Maduro, Broitman-Maduro, Mengarelli, & Rothman, 2007).  Hutter and Schnabel’s (1994) experiment found that removal of the MS blastomere from the developmental process prior to the second Notch signaling interaction results in the failure to produce pharyngeal cells.  Furthermore, inactivation of the MS daughters after the second Notch signaling interaction results in the normal formation of the anterior pharynx but it hinders the development of the posterior pharynx (Good et al., 2004).  The removal of the EMS, which gives rise to the MS blastomere, after the first Notch signaling interaction consequently leads to neither the anterior pharyngeal cell formation nor the posterior pharyngeal cell development.  These results highlight the importance of the MS blastomere and the vital role it plays in the development of C. elegans pharyngeal muscle cells.  
As mentioned previously, the pha-4 gene, which determines the identity of the pharyngeal components, is one of the key genes that regulate C. elegans pharyngeal development.  The pha-4 gene is activated during the 44-cell stage due to the expression of the tbx-37 and tbx-38 transcriptional factors and the activation of lag-1, which is induced due to the Notch signaling interaction between the ABa descendants and the MS blastomere.  When pha-4 gene is activated, it activates different genes at different time intervals (Good et al., 2004).  Mango and Lambie, and Kimble (1994) found that inactivation of the pha-4 gene or the loss of function of the pha-4 gene in mutant worms leads to the suppression of C. elegans pharyngeal development.  In other words, C. elegans pharynx does not form or develop when the pha-4 gene is not activated.  However, Arnone and his colleagues found that mutants with an ectopic pha-4 gene expression develop extra pharyngeal cells (Mango, Lambie, & Kimble, 1994).  Strangely, embryos with mutant tbx-37 and tbx-38 transcriptional factors have been found to express the pha-4 gene in intestinal and rectal cells, yet many did not demonstrate pharyngeal cell formation (Good et al., 2004).  These findings suggest that the pha-4 gene expression is important for the formation of pharynx but it still does not singlehandedly explain how pharyngeal cells, such as muscle cells, are directed to take on their specific fate.  Moreover, external factors that govern pharyngeal muscle activity still need to be explained.
The NK-2 family homeobox gene ceh-22 is linked to pharyngeal muscle formation.  It is also thought to govern the development of the heart in other higher organisms.  The ceh-22 gene, which is activated by the pha-4 gene, is the earliest gene known to be expressed in the pharyngeal muscle cell development and therefore, it is closely associated with other genes that play a role in specifying pharyngeal muscle cell fate (Vilimas, Abraham, & Okkema, 2004).  The ceh-22 gene activates the myo-2 gene, which is responsible for producing pharyngeal-muscle-specific myosin protein (Okkema et al., 1997; Vilimas et al., 2004).  Okkema et al. (1997) experimentally found that the ceh-22 gene function loss leads to a weak, thinner, and less distinct pharynx than observed in wild type nematodes.  The malformed pharynx resembles the pharyngeal phenotype of worms with a defective feeding pharyngeal phenotype because of the abnormal pharyngeal musculature (Okkema et al., 1997).  However, pharyngeal muscle cells are still present in mutants with a ceh-22 gene mutation, which suggests that there might be other factors contributing to the anterior pharyngeal muscle formation.  Smith and Mango (2007) found that tbx-2 is another major player in the anterior pharyngeal muscle fate specification.  Tbx-2 shows a similar phenotypic result as the phenotype of the ceh-22 mutant worms.  In fact, the worms with an inhibited tbx-2 or a loss of function of tbx-2 have little or no anterior pharyngeal muscle cells but these worms still possess an intact posterior pharynx (Smith & Mango, 2007).  Smith and Mango’s research findings suggest that proper coordination of the tbx-2 and pha-4 gene is essential for the successful specification of pharyngeal muscle fate.  However, research results of Vilimas et al. (2004) suggest that an enhancer sequence plus pha-4 gene is crucial for the specification of pharyngeal muscle cell fate.  It appears that the pha-4 gene is a vital gene for the determination of pharyngeal muscle fate and it works synchronously with other genes, such as the tbx-2.  There might be a chance that the pha-4 gene might utilize more than one pathway for anterior pharyngeal muscle formation.

The Formation of the Posterior of Pharynx
The development of the posterior of the pharynx does not depend on the intercellular interactions of glp-1/Notch signaling like that of the anterior pharynx; it rather depends on the mesodermal precursor MS cells that utilize a Notch-independent pathway (Bowerman et al., 1992; Maduro, Kasmir, Zhu, & Rothman, 2005).  At the 4-8 cell stage, the endomesodermal precursor EMS lineage receives signals from two maternal genes, skn-1 and pop-1, to develop pharyngeal cells.  At this stage the EMS blastomere cells are specified by bZIP/homeodomain transcription factor skn-1 that encodes bZIP-related transcriptional factors.  More specifically, skn-1 specifies the two daughters of EMS blastomere, the anterior daughter MS, which primarily form mesodermal cells and the posterior daughter E, which is endodermal precursor and forms the entire intestine (Broitman-Maduro et al., 2009; Maduro et al., 2005), through EMS blastomere by activating med-1 and med-2 transcriptional factors.  Med-1 and med-2 transcriptional factors are essential for activation of mesodermal identity genes that specify MS blastomere.  Moreover, the activation of med-1 and med-2 also activates a new T-box transcriptional factor called tbx-35, which in turn is thought to be involved in the activation of organ identity gene pha-4 in MS blastomere (Bowerman et al., 1992) (Figure 5).  This is due to tbx-35 mutant worms failing to develop the posterior pharynx.  A recent experiment (Broitman-Maduro et al., 2009) revealed that a NK-2 class homeobox gene ceh-51 is a direct target of TBX-35, where tbx-35 activates ceh-51, and both then activate genes of interest in MS blastomere development, because the removal of tbx-35 and ceh-51 together leads to similar results as does the removal of med-1 and med-2, where MS derived tissues are greatly decreased.  Also, ablation of skn-1 results in EMS descendents adopting a cousin of EMS blastomere fate, the C blastomere, as well as resulting in the total absence of pharynx because C blastomere does not produce Notch ligands to induce ABa (to form posterior pharyngeal cells) as well as the EMS (to form the posterior pharyngeal cells) (Lin, K. T., Broitman-Maduro, G., Hung, W. W., Cervantes, S., & Maduro, M. F., 2009).  The C blastomere intern leads to muscle tissue, hypodermis, and neurons, yet not any posterior pharyngeal cells.  Since the MS blastomere is also involved in the signaling of the anterior pharyngeal cellsspecification, it also hinders the formation of the anterior pharyngeal cells. 

Figure 5: Pharyngeal cell signaling pathways in its development: Genes currently known to be involved in AB descendents (brown) and P1 descendents (blue) descendents to activate organ identity gene pha-4, which leads to the formation of the anterior and posterior pharyngeal cell fate.  Lines indicate cell divisions.  Blue color indicates association to the posterior pharynx and brown color the association to the anterior color.  Genes colored orange are identified to be more specific to the anterior pharyngeal development.  Genes colored yellow are identified to the more posterior pharyngeal cell, while genes colored in green have been identified to have a role in both anterior and posterior pharyngeal cell development.  This was adapted from (Charron, 2010; Ferrier, 2009) and also utilizes the findings of (Bowerman et al., 1992; Broitman-Maduro et al., 2006; Broitman-Maduro et al., 2009; Good et al., 2004; Labouesse & Mango, 1999; Lin et al., 1995; Maduro et al., 2005; Maduro et al., 2007; Maduro, 2009; Mango, 2007; Neves & Priess, 2005; Priess, 2005; Smith & Mango, 2007; Lin, K. T., Broitman-Maduro, G., Hung, W. W., Cervantes, S., & Maduro, M. F., 2009)

Similarly to skn-1, pop-1 plays an essential role in the EMS blastomere specification.  Pop-1 specifies the anterior sister cell fate, because during the development, when cells are divided and differentiated along the anterior-posterior axis, it is observed that pop-1 is being more commonly expressed in the anterior sister cells (Labouesse & Mango, 1999).  Interestingly, EMS is one such cell that divides anterior-posterior, where the anterior cell is the MS that expresses pop-1.  The expression of pop-1 at this point here then inhibits genes responsible for the E blastomere fate (Broitman-Maduro, Lin, Hung, & Maduro, 2006).  However, pop-1 loss of function, results in MS blastomere mis-specification and leads to E-like blastomere cell fate, because pop-1 usually inhibits the activity of endoderm promoting genes end-1 and end-3 in MS blastomere that are responsible for the endoderm development (Broitman-Maduro et al., 2009; Lin, K. T., Broitman-Maduro, G., Hung, W. W., Cervantes, S., & Maduro, M. F., 2009).  As a result, when pop-1 is inactive, the formation of endoderm tissue fate is promoted instead of posterior pharyngeal cell fate (Lin et al., 1995; Maduro et al., 2005).  However, the repression of endodermal fate by pop-1 in E blastomere is overcome by Wnt/MAPK signaling that results in phosphorylation and export of pop-1 from the E nucleus (Maduro et al., 2005).  In short, posterior pharynx formation depends on med-1 and med-2 transcriptional factors (which are activated by skn-1) inducing the MS blastomere fate through activation of tbx-35, that in turn activates pha-4 to develop the posterior pharynx. 

Differentiation and Cell Fate: How a single cell develops into a multicellular organism?
A fundamental question in developmental biology is how can a single cell give rise to countless specialized cells with particular functions that eventually make up an individual.  Upon completion of fertilization, which is marked by the fusion of a sperm into an egg, the development of multicellular organisms proceeds through a series of mitotic divisions called cleavage (Gilbert, 2006).  During cleavage, the egg cytoplasm is continuously divided into many smaller and nucleated cells called blastomere, whereby the cytoplasmic volume remains unchanged (Gilbert, 2006).  As a consequence, the cytoplasmic components are distributed unevenly into blastomeres.  Moreover, these unevenly distributed cytoplasmic components are often transcriptional factors that control the activation or repression of specific genes in the blastomeres that they are acquired.  As a result, distinct groups of specific cells with specific developmental goals or programs arise when different nuclei in a different blastomere are exposed to these factors (Gilbert, 2006).  These determined groups of cells eventually become specialized parts of an animal, like the C. elegans pharynx, and together make up an entire organism.
In general, the cell fate of an organism is determined by several factors, such as contribution of maternal RNA, zygotic gene regulation, intercellular signaling, and the position of the cell in relation to its neighboring cells (also known as conditional specification) (Gilbert, 2006).  The process in multicellular organisms, like C. elegans, begins with the zygote utilizing maternal RNA to provide initial instructions for the specialization and differentiation of subsequent cells, such as AB and P1.  The AB and P1 then use zygotic gene regulation, otherwise known as differential gene expression, to continuously restrict cell fate during embryogenesis (Gilbert, 2006).  It is worth noting that the C. elegans pharynx development exhibits both autonomous (P1 blastomere dependent on Skn-1 gene) and conditional (AB blastomere dependent on Glp-1 signaling) specification (Gilbert, 2006).  This was observed when the two blastomeres were experimentally separated, where P1 developed normally all the posterior pharyngeal cells without the presence of the AB blastomere, whereas the AB blastomere only developed a fraction of the anterior pharyngeal cells (Priess & Thomson, 1987).  As cell division is continuous, the intercellular signaling, as well as the position of cells, is crucial for genetic regulators to fulfill their role in determining cell fate.  Together, maternal RNA, zygotic gene expression, intercellular signaling, and the placement of the cell during development influence each cell’s fate (Gilbert, 2006).

Morphogenesis
The development of a multicellular organism is indeed a complex process, which depends on various components and pathways to come together and work properly.  One of these processes, which is vital for the development of an organism, is called morphogenesis.  Morphogenesis is a process that is marked by cellular movement and ensures the proper differentiation and growth of specialized tissues and organs (Gilbert, 2006; Seydoux & Greenwald, 1989).  The precise regulation of cell migration and shape are essential for the formation of the three-dimensional structures of tissues and organs (Portereiko & Mango, 2001).  The C. elegans pharynx morphogenesis is initiated by the end of gastrulating when cell division is nearly complete.  At this stage, the first recognizable pharyngeal cells, primordium cells that are attached to each other by adherence junctions, are visible as a ball of cells attached to the midgut cells in the C. elegans embryo also by the adherence junctions.  The pharyngeal primordium, however, is not attached to the buccal cavity (oral cavity) at this point.  Thus, similarly to tubulogenesis of heart, kidneys, and digestive tract, these cells shift in position and orientation to form a linear tube that connects the digestive tract to the exterior of the embryo, and therefore, forming a linear tube that is connected anteriorly by epithelium to the buccal cavity and posteriorly to the midgut (Portereiko & Mango, 2001). 
The C. elegans pharyngeal morphogenesis is marked by a ball of primordium cells, which have to elongate and develop into a narrow tube of pharyngeal cells that are attached to each other by adherens junctions (Albertson & Thomson, 1976).  Pharyngeal morphogenesis can be divided into three distinct stages.  The first stage is called reorientation stage, in which most anterior pharyngeal epithelial cells rotate and rearrange their position and alter their polarity.  This leads to alteration of pharyngeal morphology from cyst to a short-linear tube and the alignment of the pharyngeal epithelial cells with the arcade cells (Portereiko & Mango, 2001).  The second stage of pharyngeal morphogenesis is coined as epithilization.  In this stage, the epidermis and digestive tract develop a continuous epithelium due to the formation of the buccal cavity adherens junctions that connect the buccal cavity to the pharynx and epidermis (Portereiko & Mango, 2001).  Finally, the third stage of pharyngeal morphogenesis is known as the contraction stage.  In this final stage, the pharynx, buccal cavity, and epidermis undergo a contraction that brings them closer in proximity.  This is as a result of movement of the pharynx anteriorly and the epidermis of the mouth posteriorly (Portereiko & Mango, 2001).  In sum, during pharyngeal mophogenesis, a ball of cells undergoes a thorough reorientation, epithilization, and contraction in order to become a functional bi-lobed organ that we know as the pharynx. 
Morphogenesis goes hand-in-hand with cell adhesion.  Cell adhesion molecules are essential for the cells to adhere to each other during morphogenesis, because they provide cells with a stable environment and allow them to migrate and thereby form three dimensional structures, like the C. elegans pharynx (Cox & Hardin, 2004; Gilbert, 2006; Gumbiner, 1996).  Also, cell adhesion molecules, such as integrins, a family of transmembrane receptors that promote cell-cell and cell-matrix adhesion, play a crucial role in promoting morphogenesis by causing neighboring cells to initiate cell migration (Hillis & Flapan, 1998; Huveneers et al., 2007; Huveneers, Truong, & Danen, 2007).  In addition to cell migration, integrins also play an essential role in cell fate, differentiation, proliferation, and programmed cell death (apoptosis).  The improper morphogenesis along with improper functioning cell adhesion complexes, such as integrins, can lead to a number of diseases, like cancer, rheumatoid arthritis (inflammatory disorders), inflammatory bowel disease, and asthma, as well as thrombosis (cardiovascular disease) (Huveneers et al., 2007).
There are many factors that are observed to alter C. elegans pharynx morphogenesis.  It has been observed that the loss of arcade cells leads to the failure of the contraction stage of the morphogenesis.  Furthermore, there are many genes seen to affect pharynx morphogenesis when their functions are experimentally altered.  For example, an ETS-domain transcriptional factor homologue, ast-1, loss of function results in pharynx unattached embryo with inability to feed (Mango, 2007; Schmid, Schwarz, & Hutter, 2006).  Depletion of another gene called pha-2, leads to abnormally thick pharynx (Mango, 2007; Mörck, Rauthan, Wågberg, & Pilon, 2004).  The eya-1 gene mutation leads to the worms’ death at L1 or L2 stage with thin pharynx phenotypes and feeding defects (Daniels, 2007; Furuya, Qadota, Chisholm, & Sugimoto, 2005; Mango, 2007).  Finally but not limited to, sma-1 mutants (a gene that is homologous to BH-spectrin and important for the elongation of pharynx) are viable but morphologically exhibit short pharynx and short bodies (Mango, 2007; McKeown, Praitis, & Austin, 1998).  As can be seen, morphogenesis of pharyngeal cells can be affected by many factors and understanding of pharyngeal morphogenesis in C. elegans, can lead us to a better understanding of morphogenesis in higher organisms such as humans.

Organogenesis
Organogenesis refers to the time during embryogenesis when the organs are being developed (Gilbert, 2006).  Organs are vital structures composed of many different cell types and tissues that are spatially and functionally organized into a unit to sustain a life.  Therefore, it is vital for this process to take place correctly because malformed organs can lead to an organism’s death.  Formation of an organ, such as the C. elegans pharynx, is a complex process, which involves activation and deactivation of many genes.  For a group of cells to develop into a functional organ, they have to undergo proper cell-to-cell communication to differentiate and acquire a proper fate, followed by and assembly into tissue through appropriate morphogenesis (Gilbert, 2006; Mango, 2007).  In sum, strictly controlled processes, such as differentiation and cell commitment, intercellular signaling, as well as morphogenesis are required for a functional organ, like the C. eleganspharynx, to develop.

Gap in knowledge
Although a great amount of information has been learned about the C. elegans pharynx development, a wealth of knowledge still remains to be uncovered about the functions and mechanisms of genes that are involved in cell fate specification and morphogenesis of pharyngeal muscle cell development.  Therefore, the goal of this research was to understand the genetic and molecular mechanisms involved in pharyngeal muscle development. 
Our lab had previously induced 265 point mutation in C. elegans pharynx using ethylmethanesulfonate (EMS), which is a technique that introduces point mutation in DNA, to study and identify genes that are vital to the pharynx as a whole and in particular to the posterior pharyngeal cell development. Furthermore, our lab has previously completed a genetic screen for worms with abnormal pharynx muscle morphology induced my EMS. The worms have an integrated myosin heavy chain structural gene with green fluorescent protein (myo-2::GFP) reporter gene, which enables rapid identification of worms with misshapen or missing pharynx.  We have screened about 10,000 haploid genomes and identified close to 200 mutant lines.  We are now focusing on two major classes of mutants: ones with a short, wide pharynx and others with amorphously shaped pharynx muscle cells. 
Twenty of these mutants manifested short and wide blunt pharynges (Ferrier, 2008).  These worms most likely have mutated genes that are responsible for embryonic elongation of the pharynx.  We have mapped and located many of these mutations to certain small regions of a particular chromosome through a single nucleotide polymorphism mapping (SNP mapping).  More specifically, our lab had previously established linkages for 10 of these mutants, of which 4 of them turned out to be homozygous recessive mutants exhibiting a blunt head phenotype.  One of these blunt head phenotypes is exhibited by a strain called mutant 77 (M77), which was chosen to be the subject of this study. We have identified two mutant lines as allelic forms of sma-1, a beta-spectrin.  Many other mutant lines are not located on any of the previously described short-pharynx gene loci.  Moreover, most of these mutant lines are larval lethal.  In fact, acrylic bead feeding assays have shown that they are unable to ingest food.  Further mapping is being conducted to identify the actual genes responsible for the phenotypes. 
Currently, the subject of this thesis, the mutation resulting in the mutant phenotype observed in M77, is genetically traced.  The M77 worms exhibit a mutated short and wide pharynx with differentiated pharynx muscle cells.  We hypothesize that through the use of complementation analysis and genetic mapping we will identify the location of the gene, and through antibody staining, we will reveal the structure of the pharynx and determine the identity of the gene causing the M77 mutation.  We further hypothesize that through complementation analysis, we will genetically balance the M77 allele.