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How many cells of each cell type are there in C.elegans?

How many cells of each cell type are there in C.elegans?


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C.elegans is a very well studied organism. Of its approximately 1000 cells in the adult stage, how many of each cell type are there? The only information I could grab from the internet (wormatlas.org) is that there are about 300 neurons. What about the rest of the cells? An answer for other simple animals such as sponges, planaria, jellyfish etc., even in relative proportion of cells, would be accepted.

(I appreciate that this question is somewhat broad and unspecific: eg. are all neurons of the same type? (Yes) Are germ cells included? (No, because they vary) Please ignore such niceties.)

Edit: To clarify, the question asks for a breakdown of the cells along the lines of: Neurons 300 cells, Epithelium 200 cells, etc., Total 1000 cells


Thanks to @MattDMo comment…

There are about 17 cell types in hermaphrodites C. elegans (see here). However, it varies very much depending how you count. You might want to have a look at Sulston and White (1988).


How many cells of each cell type are there in C.elegans? - Biology

Table 1: Number of cells in selected organisms based on counting using light or electron microscopy for values smaller than 10,000, or for larger values, estimated based on average cell size and total organism size.

The fact that all organisms are built of basic units, namely cells, is one of the great revelations of biology. Even though often now taken as a triviality, it is one of the deepest insights in the history of biology and serves as a unifying principle in a field where diversity is the rule rather than the exception. But how many cells are there in a given organism and what controls this number and their size? The answer to these questions can vary for different individuals within a species and depends critically on the stage in life. Table 1 attempts to provide a feel for the range of different cell counts based upon both measurements and simple estimates. This will lead us to approach the classic conundrum: does a whale vary from a mouse mostly in the number of cells or is it the sizes of the cells themselves that confer these differences in overall body size?

Figure 1: Estimate of the number of cells in a human body based on characteristic volumes.

Figure 2: Estimate of the number of cells in an adult human divided by cell type. Each cell type in the human body is represented as a polygon with an area proportional to the number of cells. The dominant component is red blood cells. Based on data from R. Sender et al., in preparation, 2015.

Perhaps the most intriguing answer to the question of cell counts is given by the case of C. elegans, remarkable for the fact that every individual has the same cell lineage resulting in precisely 1031 cells (BNID 100582) from one individual to the next for males and 959 cells (BNID 100581) for hermaphrodites (females also capable of self fertilization). Specific knowledge of the cell inventory in C. elegans makes it possible to count the number of cellular participants in every tissue type and reaches its pinnacle in the mapping of most synaptic connections among cells of the nervous system (including the worm “brain”) where every worm contains exactly 302 neurons. These surprising regularities have made the worm an unexpected leading figure in developmental biology and neuroscience. It is also possible to track down the 131 cells (BNID 101367) that are subject to programmed cell death (apoptosis) during embryonic development. Though not examined to the same level of detail, there are other organisms besides C. elegans that have a constant number of cells and some reveal the same sort of stereotyped development with specific, deterministic lineages of all cells in the organism. Organisms that contain a fixed cell number are called eutelic. Examples include many but not all nematodes, as well as tardigrades (aka, water bears) and rotifers. Some of our closest invertebrate relatives, ascidians such as Ciona, have an apparently fixed lineage as embryos, but they do not have a fixed number of cells as adults, which arise from metamorphosis of their nearly eutelic larvae. Having a constant number of cells therefore does not seem to have any particular evolutionary origin but rather seems to be a common characteristic of rapidly developing animals with relatively small cell numbers (on the order of 1000 somatic cells).

Figure 3: Plant and organ size changes from domestication, breeding hybridization and transgenic modification. These variations are found to be mostly driven by change in cell number. Fruit size of wild and domesticated species: (A) wild relative species of pepper, Capsicum annuum cv. Chiltepin (left) and bell pepper (right) (B) wild relative species of tomato, Solanum (left), Solanum esculentum cv Giant Red (right)

In larger organisms, the cellular census is considerably more challenging. One route for making an estimate of the cellular census is to resort to estimates based upon volume as shown in Figure 1. For example, a human with a mass of ≈100 kg will have a volume of ≈10 -1 m 3 . Mammalian cells are usually in the volume range 10 3 -10 4 μm3=10 15 -10 -14 m 3 , implying that the number of cells is between ≈10 13 -10 14 which is the range quoted in the literature (BNID 102390). Though the sizes (linear dimension) of eukaryotic organisms can vary by more than 10 orders of magnitude, the size of their cells measured by the “radius”, for example, usually varies by only a factor of ten at most except for intriguing exceptions such as the cells of the nervous system and oocytes. However, the level of accuracy of estimates like those given above on the basis of volume should be viewed with a measure of skepticism as can be easily seen by considering your recent blood test results. The normal red blood cell count is 4-6 million such cells per microliter. With about 5 liters of blood in an adult this results in an estimate of 3吆 13 such cells rushing about in your blood stream, already for this cell type alone as many as the total number of cells in a human body we estimated using volume arguments. The disagreement with the estimate above results from the fact that red blood cells are much smaller than the characteristic mammalian cell at about 10 2 μm 3 in volume. This shows how the above estimate should in fact be increased (and several textbooks revised). A census of the cells in the body was achieved by methodically analyzing different cell types and tissues arriving at a value of 3.7±0.8吆 13 cells in a human adult (BNID 109716). The breakdown by cell type for the major contributors is shown in Figure 2. The numerical dominance of red blood cells is visually clear. Of course we do not account for bacterial cells or other residents in our body, the number of cells composing this so called microbiota outnumber our human cells by a factor still unknown but probably closer to a hundred than to the often quoted value of ten.

Figure 4: Adipocyte number remains stable in adulthood, although significant weight loss can result in a decrease in adipocyte volume. Total adipocyte number from adult individuals (squares) was combined with previous results for children and adolescents (circles) The adipocyte number increases in childhood and adolescence. (Adapted from K. L. Spalding, Nature 453:783, 2008.)

What is the connection between organism size, cell size and cell number? Or to add some melodrama, does a whale mostly have larger cells or more cells than a mouse? In studying the large variation in fruit organ size as shown in Figure 3 it was found that the change in the number of cells is the predominant factor driving size variability. In the model plant Arabidopsis thaliana, early versus later leaves vary in total leaf area from 30 to 200 mm 2 (BNID 107043). This variation comes about as a result of a concomitant change in cell number from 20,000 to 130,000 with cell area remaining almost constant at 1600 μm 2 (BNID 107044). In contrast, in the green revolution that tripled yields of rice and wheat in the 1970’s, a major factor was the introduction of miniature strains where the smaller size makes it possible for the plant to support bigger grains without falling over. The smaller cultivars were achieved through breeding for less response to the plant hormones gibberellins that affects stem cell elongation. In this case, a decrease in cell size, not cell number, is the dominant factor, a change in the underlying biology of these plants that helps feed over a billion people.

When the ploidy of the genome is changed the cells tend to change size accordingly. For example, cells in a tetraploid salamander are twice the size of those in a diploid salamander, although the corresponding organs in the two animals have the same size. Everything fits well because the tetraploid salamander contains half as many cells as the diploid (BNID 111481).

Figure 5: Average fat cell size as a function of body fat mass. As the fat content of a person increases the average adipocyte volume initially increases almost linearly and then saturates. Thus the change in total fat among humans can be attributed mostly to larger cells of a similar number and at more extreme disparities also to change in the number of fat cells. (Adapted from K. L. Spalding, Nature 453:783, 2008.)


1. Introduction

There are two sexes in C. elegans , hermaphrodite and male. Hermaphrodites are basically females that produce a small number of sperm that can fertilize their own oocytes. They are also cross-fertile with males. Males are smaller than hermaphrodites and produce only sperm. There are many sex-specific differences between males and hermaphrodites. Although these differences affect most tissues, the basic body plan and many of its structures are identical (Sulston et al., 1980 Sulston and Horvitz, 1977). In fact, about 650 cells seem to be the same in both sexes, out of a total of 959 somatic nuclei in hermaphrodites, and 1031 somatic nuclei in males. Most structures required for mating or reproduction are sexually dimorphic. For example, the hermaphrodite vulva is a reproductive structure that functions to allow eggs to be laid and male sperm to be deposited. Males develop highly specialized reproductive structures in the tail that allow it to find the hermaphrodite vulva and deposit sperm (Figure 1). Furthermore, the gonads of each sex have different structures and functions. Finally, sexually dimorphic neurons and muscles develop in each sex to control the movements of these mating structures.

Figure 1. Major differences between the sexes are found in the posterior and the ventral body. In the male, cells of the posterior lateral seam generate the sensory rays, instead of alae. hyp cells of the tail tip reorganize to form a blunt tail morphology. The hypodermis (or lateral seam) secretes the cuticular fan in which the sensory rays are embedded. Internally, rectal epithelial cells (B, F, and U) divide to produce the proctodeal and cloacal lining of the reproductive tract, replacing the rectum. The B lineage also generates the support cells (and neurons) of the spicules. Ventrally, hypodermal P cells in the posterior generate the hook. The rectal epithelial cell Y produces the support cells (and neurons) of the PCS, which lies posterior to the hook. In hermaphrodites, P cells in the mid-body make the vulval epithelium. Reprinted with permission from Wormatlas (http://www.wormatlas.org).

Each of these structures is generated by sex-specific cell lineages. For example, certain P cells divide to generate the vulva in hermaphrodites, while other P cells divide to generate portions of the mating apparatus of the male tail (Figure 2). Other male specific blast cells that do not divide in hermaphrodites generate additional mating structures in the tail. In addition, the M cell divides in hermaphrodites to generate the vulval muscles that control the movements of the vulva. In males, the M cell divides to generate sex muscles that control movements of the tail (Figure 3). Interestingly, almost all of these sexually dimorphic structures are made during larval development by the sex-specific divisions of just 16 blast cells (Figure 4).

Figure 2. Hermaphrodite and male P lineages. Development of the ventral nervous systems. Dotted lines indicate the times nuclei migrate into the ventral cord. Divisions are anterior-posterior unless otherwise indicated. Modified and reprinted from Sulston, J. E., and Horvitz, H. R. (1977), Copyright (1977), with permission from Elsevier. Control-click or right-click to see larger image.

Figure 3. Hermaphrodite and male M lineages. Divisions are anterior-posterior unless otherwise indicated. bm, body muscle cc, coelomocyte um1, type 1 uterine muscle um2, type 2 uterine muscle vm1 type 1 vulval muscle vm2, type 2 vulval muscle. Modified and reprinted from Sulston, J. E., and Horvitz, H. R. (1977), Copyright (1977), with permission from Elsevier. Control-click or right-click to see larger image.

Figure 4. Male and hermaphrodite L1 larvae. At hatching, male and hermaphrodite larvae are anatomically identical apart from a few cells (blue labels). Most sex-specific tissues of the adult are formed from the descendents of blast cells (black labels), that are present in both sexes but express different fates or lineages in each sex. Reprinted with permission from Wormatlas (http://www.wormatlas.org).

Since most of the cell fate specifications that occur in hermaphrodites also occur in males, the focus of this chapter will be on those that only occur in hermaphrodites. Thus, I will include the cell fate decisions that affect the HSN neurons, ventral hypodermal P cells, lateral hypodermal cells V5, V6, and T as well as the mesodermal M, Z1, and Z4 cells. Even the intestinal cells have sex-specific fates (Figure 5). Aspects of many of these cell fate decisions are covered in more detail in other chapters, which will be referred to where appropriate.

Figure 5. Sexually dimorphic hermaphrodite cells. Nuclear positions of sexually dimorphic hermaphrodite cells discussed in this chapter. Green nuclei are ectodermal, red nuclei are mesodermal. Individual nuclei of intestine are not shown, brown indicates endoderm. Anterior is to the left.


Scales and Rulers

To find out, the scientists measured the kinetic properties of an important polarizing protein in normal C. elegans embryos and in embryos whose sizes they had genetically manipulated. As expected, the protein’s diffusion rate and other qualities did not change, even when the cells got bigger or smaller. Instead, the patterning system had its own intrinsic scale, one that didn’t adjust to the overall size of the cell.

By controlling the sizes of the initial embryos, the team was then able to show that there was a minimum size threshold for the P lineage cells, below which they could not set up the polarization pattern. Those smaller cells lost the ability to polarize after just three cell divisions, not four. “Just by manipulating the size of the embryo, we’ve taken a cell that normally would be able to polarize and divide asymmetrically and turned it into a cell that doesn’t polarize and divides symmetrically,” Goehring said.

Moreover, a perusal of previous research revealed that two other worm species have one extra asymmetric division in their P lineage. Their P lineage cells tend to start bigger (and stay bigger) than those of the early C. elegans embryo, in keeping with Goehring’s theory. Whether the same mechanism is truly at work in those species remains to be tested, but it doesn’t seem like a coincidence.

Cells have seemingly evolved to take advantage of the intrinsic limitations of their patterning process — using it as a ruler of sorts — to determine whether to become germ cells. “The specification [of the germ cells] is a kind of self-organized property of the patterning system,” Howard said.

And that’s a “genuinely interesting” way to think about the system, said Timothy Saunders, a biophysicist at the Mechanobiology Institute of the National University of Singapore who was not involved in the study. “This idea, that by just simply making things smaller you can naturally switch the type of division, is very neat.”


Scientists launch project to map every cell in human body

Once completed, the "Human Cell Atlas" could revolutionize how diseases are diagnosed and treated, according to the Wellcome Trust Sanger Institute, one of the meeting organizers.

As ambitious in scope as the Human Genome Project -- which cataloged the first full human DNA sequence -- the Human Cell Atlas aims to chart the types and properties of all human cells to build a reference map of the human body, according to researchers involved in the project.

"The cell is the key to understanding the biology of health and disease, but we are currently limited in our understanding of how cells differ across each organ, or even how many cell types there are in the body," said Sarah Teichmann, head of cellular genetics at the Sanger Institute.

This initiative is the beginning of a new era of cellular understanding, she said. "We will discover new cell types, find how cells change across time, during development and disease, and gain a better understanding of biology," she said in an institute news release.

The result will be a valuable, free resource for biomedical science researchers -- whether they're studying human development or the progression of diseases such as asthma, Alzheimer's and cancer, she and her colleagues said.

The London meeting will help establish the first phase of the initiative.

For years, scientists only had the microscope to broaden their knowledge of cells. But recent high-tech advances in the field of single-cell genomics has made it possible to separate individual cells from different tissues and organs, and measure important molecules from each of them, the researchers explained.

"We now have the tools to understand what we are composed of, which allows us to learn how our bodies work, and uncover how all these elements malfunction in disease," said Aviv Regev, faculty chair at the Broad Institute of MIT and Harvard. The Broad Institute was also involved in organizing the meeting.

A successful description of all the cells in the healthy human body will affect almost every aspect of biology and medicine in the decades to come, Regev predicted.

"By creating this atlas through an open, international effort, we are building a new research tool for the whole community," she said.

Arizona State University has more about human cells.

Copyright © 2016 HealthDay. All rights reserved.


Conclusion

Our work identifies a divergence in the ability of autophagy to clear aggregates in different tissues. As activation of autophagy is a promising therapeutic strategy for protein aggregation diseases, the vulnerability of muscle cells in our study highlights the need for a more nuanced understanding of how autophagy integrates with cellular physiology.

Importantly, the finding that dramatic differences in polyglutamine aggregation can be caused by physiological-level differences in the autophagic response, encoded in wild-type genomes, supports the use of natural genetic variation in model organisms to interrogate pathways that confer protection or susceptibility in protein aggregation diseases.


Murray Lab

The goal of the Murray laboratory is to understand how genomes control animal development. Animals, including humans, consist of many highly specialized cell types which must be generated in the correct number and at the right time and place to allow the organism to function correctly. Most of our understanding of this process comes from studies that focus on individual cell types or tissues, limiting our understanding of how regulators and mechanisms apply across all cells in an organism. The Murray laboratory develops and uses whole-organism live-cell imaging, and genomics methods, combined with classical genetics, to study gene regulation across the entire embryo. We use the nematode worm Caenorhabditis elegans as a model for many of our studies because it shares most of its cell types and regulatory mechanisms with humans, but is much easier to study.

A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution.

3D projection of single cell RNA expression data from C. elegans embryos

RESULTS

We profiled the transcriptomes of 86,024 single cells from C. elegans embryos at developmental stages ranging from gastrulation to terminal cell differentiation. Using computational methods, gene expression patterns from the literature, and gene expression data obtained from three-dimensional (3D) movies of fluorescent reporter lines, we mapped each single-cell transcriptome to its corresponding position in the known C. elegans cell lineage tree. In total, we identified 502 distinct terminal and preterminal cell types, which correspond to 1068 individual branches of the lineage tree. We computed a transcriptional profile for each detected cell type and determined the gene expression differences between mother and daughter cells, and between sister cells, for >200 cell division events in the lineage.

Analyzing these data, we find that:

1) A cell’s lineage history and its transcriptome are transiently correlated. This correlation increases from middle to late gastrulation, then falls substantially as cells adopt their terminal fates.

2) Genes that distinguish sister cells are often first coexpressed in the parent and then selectively retained in one daughter but not the other. This phenomenon, known as “multilineage priming,” is notably prevalent throughout the C. elegans lineage.

3) Most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state, with little or no residual signature of their lineage identity.

4) In many cases, purely computational reconstruction of developmental trajectories from the single-cell transcriptomic data does not accurately reproduce the known cell lineage. Marker genes known to be expressed in specific lineages were critical for correct annotation. This is particularly evident for lineages in which gene expression changes rapidly.

CONCLUSIONS

Our dataset defines the succession of gene expression changes associated with almost every cell division in an animal’s embryonic cell lineage. It provides an extensive resource that will guide future investigations of gene regulation and cell fate decisions in C. elegans. It can also serve as a benchmark dataset that will facilitate rigorous evaluation of computational methods for reconstructing cell lineages from sc-RNA-seq data.

Want to join us?

Contact John to apply for a postdoctoral position. We are affiliated with several graduate programs at Penn including:


RESULTS

Elt-5 and -6 are adjacent genes that encode similar GATA factors

In an effort to learn how the C. elegans epidermis becomes patterned into the three major epidermal cell types during embryogenesis, we have attempted to identify factors that impart seam cell-specific identity (Terns et al., 1997). Because GATA factors, namely ELT-1 and -3, are involved in other aspects of epidermal development, we examined several GATA factor-encoding genes predicted from the C. elegans genomic sequence (C. elegans Sequencing Consortium, 1998) for a possible role in embryonic seam cell development. We found that RNA-mediated interference (RNAi) of a GATA factor-encoding gene we have named elt-5 (erythroid-like transcription factor 5) results in penetrant embryonic and early larval lethality and causes morphological defects that include the lack or malformation of seam-specific cuticular specializations, called alae. This observation led us to investigate the role of elt-5 and its paralog, elt-6 in seam cell development.

elt-5 and elt-6 are adjacent genes encoding single-finger GATA factors. Their encoded DNA binding domains are 76% identical and are ∼60% identical to those in other GATA factors (Fig. 1). As is typically the case for GATA factors, ELT-5 and -6 are not significantly similar to the other GATA factors outside the DNA-binding domains. However, the two proteins are 46% identical overall, implying that one of the genes arose by duplication of the other. The putative polyadenylation site of the upstream gene, elt-5, and the trans-splice site of the downstream gene, elt-6, are separated by only ∼130 base pairs, characteristic of genes that reside on the same operon (Blumenthal and Steward, 1997). However, attempts to determine whether the two genes indeed form an operon have not produced conclusive evidence. As most, if not all, downstream genes are trans-spliced to the SL2 leader (Spieth et al., 1993 Zorio et al., 1994), we looked for, but failed to find, evidence for SL2 trans-splicing of transcripts from the downstream gene, elt-6 (see Materials and Methods). All seven elt-6 cDNA clones examined were either trans-spliced to the SL1 leader or were not trans-spliced. This suggests that elt-6 is often transcribed from its own transcription initiation site near the SL1 splice site rather than co-transcribed with elt-5 as a dicistronic transcript. This interpretation is consistent with data obtained with a reporter construct (see below). However, based on the effects of elt-5 dsRNA on reporter constructs, it seems likely that at least a fraction of mature elt-6 message is generated from elt-5/elt-6 dicistronic transcripts (see below).

Interference of elt-5 and -6 function leads to defects in seam cell development in embryos and larvae

We used the technique of RNAi (Fire et al., 1998 Guo and Kemphues, 1995) to assess the developmental function of elt-5 and -6. We found that nearly all (90/95) progeny of hermaphrodites injected with high levels of elt-5 dsRNA (see Materials and Methods) arrest late in embryogenesis (pretzel stage) or as early L1 larvae. The arrested L1 larvae are invariably uncoordinated (Unc phenotype), lumpy (Lpy phenotype) and slightly dumpy (Dpy phenotype), suggesting defects in epidermal development (Fig. 2B). In addition, the entire buccal capsule, the cuticular structure of the mouth (Wright and Thomson, 1981), invariably fails to attach to the anteriormost region of the head (‘pharynx unattached’ or Pun phenotype Fig. 2B), suggesting defects in the buccal epidermis. A small fraction (5/95) of embryos arrest earlier, apparently with ruptures at the head or ventral midline (not shown).

Injection of elt-6 dsRNA at high levels did not produce any observable phenotype, and co-injection of both elt-5 and elt-6 dsRNAs at high levels did not result in an enhanced phenotype compared with elt-5 dsRNA alone. These results, however, do not necessarily indicate that the observed phenotypes are due to elimination of elt-5 function alone. In fact, elt-5 dsRNA affects expression of both elt-5 and -6 in seam cells (see below), and the observed phenotypes of elt-5(RNAi) animals may arise from inhibition of both genes or of elt-5 alone. For simplicity, we will use the notation elt-5/6 to refer to the function of either the elt-5 gene alone or of both the elt-5 and -6 genes. Based on its effects on reporter gene constructs, elt-5 dsRNA at high levels appears to abolish its function (as well as elt-6 function in some tissue types see below) thus, these phenotypes are likely to reflect a strong loss-of-function or null phenotype.

To investigate a possible post-embryonic role for elt-5, we injected hermaphrodites with lower levels of elt-5 dsRNA. Such injections resulted in a mixture of weakly and strongly affected progeny. Weakly affected larvae appeared normal at hatching they were neither Lpy nor Unc, and their buccal capsules were properly attached. Many of these larvae, however, became lethargic and sickly at later stages, were molting defective, and arrested at various stages of larval development. A small fraction of the weakly affected larvae also showed other gross morphological abnormalities that were suggestive of epidermal defects, including Roller (Rol) and protruding vulva (Pvl) phenotypes (not shown). The post-embryonic developmental defects were examined in more detail, as described later. All elt-5(RNAi) animals described in the remainder of this paper were obtained from mothers injected with high levels of elt-5 dsRNA.

The gross phenotypes we observed suggested defects in epidermal structure and/or development. To characterize the epidermis in elt-5(RNAi) embryos, we visualized their epithelial adherens junctions with monoclonal antibody MH27 (Priess and Hirsh, 1986 Waterston, 1988). In a wild-type embryo, all epidermal cells, including the row of ten lateral seam cells on each side, are clearly outlined by MH27 staining (Fig. 2C). In contrast, although most of the epidermal pattern appeared normal in elt-5(RNAi) embryos, the rows of seam cells often showed gaps in MH27 staining (Fig. 2D). In addition, seam cells were occasionally displaced from the linear row of lateral cells. For example, Fig. 2F shows an elt-5(RNAi) embryo in which a seam cell, V1 (asterisk), is ventrally misplaced such that neighboring seam cells, H2 and V2, contact each other (compare with Fig. 2E). The misalignment and gaps in the pattern were never observed in P cells.

There are at least four possible explanations for the gaps in the seam rows seen in elt-5(RNAi): (1) seam cells are misspecified as non-epidermal cells at birth (2) seam cells are misspecified as epidermal syncytial cells at birth, leading them to fuse with other syncytial epidermal cells (3) seam cells, although correctly specified initially, lose their identity and later adopt a syncytial-type identity or (4) seam cells retain their seam identity, but fusion is misregulated. (For simplicity, cells that normally become seam cells in wild-type will be called seam cells, irrespective of their ultimate identity in elt-5(RNAi) animals.) In an effort to distinguish between these possibilities, we examined the epidermal pattern over time using the JAM-1::GFP marker, which reveals the MH27 adherens junction pattern in living embryos (Mohler et al., 1998). These studies demonstrated that the gaps in the seam row probably result from fusion of existing seam cells with the surrounding epidermal syncytium. In wild-type embryos, most dorsal and ventral syncytial cells complete their fusion between the 1.5- and twofold stage of elongation (Podbilewicz and White, 1994). In elt-5(RNAi) embryos at the same stages, only occasional lateral cells lacked JAM-1::GFP expression. By hatching, however, many lateral cells (32%, n=61 larvae) lacked the adherens junction marker. This progressive disappearance of the adherens junction marker from the lateral row results from cell fusion we were able to observe ongoing dissolution of the adherens junctions between individual lateral cells and the adjacent epidermal syncytium as they were caught in the act of fusion (see Fig. 4H, arrowhead). These ongoing fusions were observed in some cases as late as in newly hatched animals.

The relatively late onset of fusion (i.e. after the time that the normal epidermal syncytial cells fuse) (Podbilewicz and White, 1994) suggests that many seam cells that ultimately fuse with surrounding syncytia are initially specified correctly in elt-5(RNAi) embryos. This notion was supported by observing expression of SCM, a seam cell-specific marker (Terns et al., 1997). In wild-type embryos, SCM is expressed in all seam cells from the twofold stage through adulthood (Fig. 2G). We found that SCM expression is visible in all seam cells of elt-5(RNAi) embryos at the threefold stage (n=52), although arrested embryos and larvae show somewhat reduced expression (not shown). These observations revealed that seam cells are not misspecified as syncytial cells at the time of their birth in elt-5(RNAi) embryos. Consistent with the view that cells that have been specified as seam cells subsequently fuse with the syncytium, we often observed several nuclei in the epidermal syncytium, surrounding the region in which a seam cell had fused, ‘ectopically’ expressing SCM, albeit at low levels (Fig. 2H, arrowheads). Presumably fusion of SCM-expressing seam cells into neighboring syncytia allows release of some GFP molecules, which are taken up by nearby syncytial nuclei.

Morphological observations indicated that seam development in elt-5(RNAi) animals is abnormal even in the seam cells that do not fuse. Seam cells normally produce the alae, bilateral ridges of specialized cuticle superjacent to the seam cells of L1 larvae, dauer larvae, and adults. Wild-type alae are clearly evident at the L1 stage as two parallel ridges running along the body on each side (Fig. 3A,E). We found that the alae in elt-5(RNAi) larvae were invariably missing or malformed: 86% of elt-5(RNAi) larvae showed no visible alae, and 14% had partial and/or defective alae (n=56). For example, Fig. 3B,F show an elt-5(RNAi) larva that lacks alae over most of its length and that contains a stretch of ala with several irregularly shaped branches. By correlating the position of missing alae with the JAM-1::GFP pattern, it was evident that seam cells failed to produce alae irrespective of whether or not they had fused (Fig. 3D).

Seam cells fail to differentiate properly and inappropriately express a non-seam marker in elt-5(RNAi) embryos

We assessed the range of seam cell characteristics that require elt-5/6 by analyzing several markers of seam-specific fate. As noted earlier, SCM, a marker of seam fate, is expressed in the seam cells of elt-5(RNAi) embryos, implying that seam-specific differentiation is initiated in these mutants. Eight genes that encode nuclear hormone receptors (NRs) are also apparently expressed exclusively in seam cells (Miyabayashi et al., 1999). We found that expression of reporters for some, but not all of these NR genes was diminished or abolished in elt-5(RNAi) embryos (Table 1). Expression of three NR reporters, nhr-75::GFP, nhr-81::GFP, and NHR-82::GFP, was undetectable in all elt-5(RNAi) embryos examined (Fig. 4A,B). In contrast, expression of two NR reporters, nhr-73::GFP and nhr-74::GFP, was only slightly affected, both in terms of the fraction of expressing animals and the level of GFP signal (Fig. 4C,D). The remaining three NR reporters, nhr-72::GFP, nhr-77::GFP, and nhr-89::GFP, gave intermediate results these were expressed much less frequently in elt-5(RNAi) embryos than in wild-type embryos, and expression was barely detectable in only a few cells for those embryos showing any expression (Fig. 4E,F).

The foregoing observations indicate that elt-5/6 is essential for many, but not all, aspects of seam cell differentiation. To investigate the possibility that they may also participate in specifying seam identity, we examined the expression of the elt-3::GFP reporter, which accurately reflects expression of endogenous ELT-3 (Gilleard et al., 1999). At the 1.5-fold stage, elt-3::GFP is expressed in all major epidermal cells except seam cells (Fig. 4G). In contrast, we found that seam cells often express elt-3::GFP ectopically in elt-5(RNAi) embryos (Fig. 4H). As most seam cells in elt-5(RNAi) embryos are still unfused at this stage, the expression of elt-3::GFP cannot be simply the consequence of seam fusion, but apparently reflects a partial transformation in fate of seam cells into non-seam cells. In nearly all (19/20) 1.5-fold stage embryos examined in detail, at least one unfused seam cell, and an overall average of 27% of the unfused seam cells, expressed elt-3::GFP. Of particular note, the V3 seam cell was the most likely to express elt-3::GFP (80% of V3s examined). However, V3 was not the most likely to fuse. One explanation for this behavior is that, of all the seam cells, V3 is the most closely related to P cells: its sister and all its cousins are P cells (Sulston et al., 1983). It is therefore possible that in the absence of elt-5/6, V3 often adopts the fate of its sister and cousins (i.e. the P cell fate) and therefore both expresses elt-3 and remains unfused.

These findings indicate that elt-5/6 is required to maintain the identity of seam cells. Its role in repressing cell fusion may reflect conversion to a syncytial epidermal fate in the absence of seam-specifying information.

Elt-5 and -6 are expressed in seam cells

To assess the expression patterns of elt-5 and -6, we created several transcriptional and translational reporter constructs (Fig. 5). An elt-5 translational fusion construct (pKK52), in which the elt-5 promoter drives expression of a GFP::ELT-5 fusion protein, shows a complex and dynamic expression pattern that can be divided into three major components. First, pKK52 expression begins at the 28-cell stage in all four granddaughters and 16 great-great granddaughters of the MS and AB founder cells, respectively (Fig. 6A) this expression continues in many, possibly all, of their descendants until around the time of hatching. Second, expression becomes more pronounced in seam cells about 1 hour after their birth. This seam expression remains strong throughout embryonic and larval development (Fig. 6B), but becomes slightly reduced in adults. Third, robust expression is also seen in several cells in the head region, at least some of which are cells in the nervous system (neurons and/or support cells), beginning at approximately the comma stage (Fig. 6B) and continuing through adulthood. For simplicity, we will refer to this component of the expression pattern as nervous system expression, although we have not determined the precise identity of these cells.

An elt-6 transcriptional reporter (pKK41) is expressed in the same groups of cells as the elt-5 translational reporter (pKK52), but the relative expression levels are different. Whereas the elt-5 reporter is strongly expressed in both seam cells and the nervous system during the comma through pretzel stages (Fig. 6B), the elt-6 reporter is strongly expressed only in the nervous system (Fig. 6E). Only weak expression of the elt-6 reporter is apparent in seam cells and in the AB and MS descendants during embryogenesis, but the seam expression becomes stronger during larval development (not shown). Strong expression of the elt-6 reporter in the nervous system continues throughout larval development (Fig. 6F).

To confirm the expression patterns obtained with the reporter constructs, we raised antibodies against peptides specific for ELT-5 and -6. For each protein, two peptides were selected from regions of little or no similarity between the two proteins. Anti-ELT-5 staining is readily detected in the nuclei of seam cells during mid- to late-embryogenesis (Fig. 6C,D). At these stages, many unidentified cells in the head region also stained, consistent with the pattern seen for the GFP reporters (Fig. 6B). This staining is eliminated in elt-5(RNAi) embryos (not shown). We have been unable to obtain consistent and reliable staining of early embryos, larvae and adults, and therefore have not confirmed the reporter expression pattern at these stages. Anti-ELT-6 staining is most readily seen in several cells in the head and is faint in seam cells (Fig. 6G,H), consistent with the GFP reporter data. elt-6 dsRNA eliminates all nuclear staining (not shown).

Monocistronic transcription of elt-6 messages and modularity of tissue-specific enhancers

As described above, despite the apparent operon-like organization of the elt-5 and -6 genes, we could not find evidence that elt-6 cDNA is trans-spliced to SL2. Previous studies have shown that some downstream genes in apparent operons are transcribed monocistronically under the control of their own promoters (Gilleard et al., 1997). To test whether elt-6 can be transcribed monocistronically, we constructed an elt-6 reporter (pKK44), which includes the unusually large (>2 kb) last intron and the last exon of elt-5 upstream of the elt-6 ATG (see Fig. 5). pKK44 is expressed strongly in some cells in the nervous system but is expressed in neither seam cells nor in early AB and MS descendants (not shown). Because pKK44 lacks the 5′ end and the first three exons of elt-5, this result indicates that elt-6 messages can be transcribed alone.

The result with pKK44 also suggests that enhancer sequences for seam, AB and MS expression are separable from those for expression in the nervous system. Indeed, we found that an elt-5 transcriptional reporter (pKK7) that includes only the 3.4 kb sequences upstream of the elt-5 ATG (see Fig. 5) is expressed in the early AB and MS lineages and in seam cells, but is not expressed in the nervous system. This pattern is complementary to the expression pattern of pKK44, demonstrating that separable enhancer regions regulate elt-5 and -6 expression in different groups of cells. Whereas enhancers for seam, AB and MS are contained in the 3.4 kb region upstream of elt-5, those for the nervous system reside in the 3.1 kb region upstream of elt-6 (perhaps within the last, large intron of elt-5).

Efficacy of RNAi and evidence for tissue-specific monocistronic versus dicistronic transcription

To determine the effectiveness and specificity of the elt-5 and -6 dsRNAs, we injected them into strains carrying various elt-5 and -6 GFP reporter genes (Table 2). Expression of an elt-5 translational fusion (pKK39) was reduced to undetectable levels in elt-5(RNAi) embryos, suggesting that RNAi results in a strong loss-of-function or null phenotype. Furthermore, although elt-6 dsRNA did not cause an observable phenotype, we confirmed that elt-6 dsRNA was also effective expression of an elt-6 translational fusion (pKK47) was eliminated by elt-6 dsRNA. In contrast, consistent with only moderate similarity between the two genes (∼60% identity overall), elt-5 dsRNA did not significantly affect expression of the elt-6 translational fusion, pKK47, which lacks the elt-5 coding region, and elt-6 dsRNA likewise did not alter expression of an elt-5 fusion (pKK39).

Previous studies have suggested that in some cases RNAi targeted against one gene in an operon can inhibit expression of another in the same operon (Bosher et al., 1999). We therefore explored the possibility that elt-5 dsRNA interferes with expression of both elt-5 and -6 and found that, indeed, elt-5 dsRNA blocks expression of the elt-6 fusion pKK41 (which contains the elt-5-coding region) in seam cells and in early AB and MS descendants, but not in the nervous system. (This probably does not relate to the relative insensitivity of the nervous system cells to RNAi (Tavernarakis et al., 2000), as we were able to eliminate expression of elt-6 in these cells using elt-6 dsRNA (data not shown).) Because pKK41 does not include any portion of elt-6, this is not due to cross-hybridization: instead, this result implies that elt-5 dsRNA eliminates expression of elt-6 in some (although not in all) cell types. However, elt-6 dsRNA did not affect expression of an elt-5 fusion (pKK39) that includes both elt-5- and elt-6-coding regions, suggesting that a fraction of the elt-5 transcripts are monocistronic. In summary, our results suggest that in seam cells and in early AB and MS descendants elt-5 is either transcribed alone or co-transcribed with elt-6, while in the nervous system elt-5 and -6 are each transcribed monocistronically (although there may be some dicistronic transcripts in the nervous system as well). Collectively, our findings suggest that the apportionment of monocistronic versus dicistronic transcription of this pair of genes is regulated in a tissue-specific manner.

ELT-6 can rescue the lethality of elt-5(RNAi) animals, revealing an apparently continuous post-embryonic requirement for elt-5

Given the strong similarity of the ELT-5 and -6 proteins, we explored the possibility that they are functionally interchangeable. To test whether elt-6 can rescue the absence of elt-5, we created a GFP::ELT-6 fusion, driven by the 3.4 kb upstream region of elt-5 (pKK47, Fig. 5), which allowed us to drive expression of ELT-6 in seam cells and AB and MS descendants even in the presence of elt-5 dsRNA. Most elt-5(RNAi) animals expressing pKK47 (52 of 57 animals carrying the construct, as assessed by GFP expression) appeared wild type at hatching, and many (48/57) grew up to be viable adults. These rescued animals appeared to develop normally with the exception that many of them lacked a vulva (not shown), as will be described in more depth in a separate publication. These results demonstrate that elt-5 and -6 are functionally interchangeable through most of development and that expression of ELT-6 in seam cells and in AB and MS descendants is sufficient to rescue elt-5(RNAi) animals to viability.

We next asked whether the elt-5(RNAi) phenotypes we observed were attributable to the activity of the gene in seam cells, in AB and MS descendants, or both. Because all seam cells are derived from the AB lineage, it is not possible to completely separate the two components. We therefore created two rescuing constructs, pKK25 and pKK49, with partially overlapping, but largely complementary expression patterns (Fig. 5). In construct pKK25, expression of a GFP::ELT-6 translational fusion is driven by the 1.2 kb upstream promoter of elt-5. This construct is expressed in AB and MS descendants from early embryogenesis until about the time of hatching and is expressed in seam cells during embryogenesis only as part of the broad AB expression pattern. By the late L1 stage, however, no GFP is detectable. We created another construct, pKK49, in which the GFP::ELT-6 fusion protein is driven by the nhr-74 promoter. This promoter, which is expressed only in seam cells from about the comma stage through adulthood (Miyabayashi et al., 1999), was chosen because it is not significantly affected by elt-5 dsRNA (Table 1). The sum of the expression patterns of the two constructs closely resembles that of pKK47, the GFP::ELT-6 fusion construct containing the 3.4 kb elt-5 promoter. Indeed, we found that most (31/44) elt-5(RNAi) embryos carrying both pKK25 and pKK49 were rescued and grew up to become fertile adults.

While each of these constructs alone was not sufficient for viability, they allowed us to assess the temporal and tissue requirements for elt-5/6. All (n=54) elt-5(RNAi) animals expressing GFP::ELT-6 only in seam cells (from construct pKK49) arrested as embryos or larvae by the early L1 stage, the stage at which elt-5(RNAi) animals normally arrest. The arrested animals showed the Pun phenotype (Fig. 7A), which is presumably the cause of lethality. However, the alae defects normally seen in elt-5(RNAi) larvae were rescued (31/33 larvae showed normal alae Fig. 7B), implying a cell-autonomous function of elt-5/6 in alae formation. In addition, the Lpy phenotype was partially rescued: the animals were only slightly lumpy, indicating that elt-5/6 activity in seam cells contributes to proper morphogenesis.

In contrast, most (36/42) elt-5(RNAi) larvae expressing GFP::ELT-6 in AB and MS descendants (from construct pKK25) developed beyond the early L1 stage, but many (32/42) had missing or malformed alae. Those that developed beyond early L1 (n=36) arrested growth by the L3-L4 molt. Of particular significance, by the time these larvae arrested, nearly all of their seam cells had fused with the surrounding epidermal syncytia (Fig. 7C,D). Some larvae (5/36) showed no visible seam cell boundaries at all, and the rest contained between one and four seam cells with distinct boundaries (overall the mean number of unfused seam cells in 36 larvae was 1.8). In addition, the arrested larvae were defective in molting: though they apparently initiated the molting process, they were unable to shed their old cuticle completely (Fig. 7E,F). Many of these were still encased in the old cuticle, and because their mouth was blocked they were unable to eat this is presumably the cause of lethality (Fig. 7E). The old cuticle was often wrapped around the body of these larvae, forming a constriction, and sometimes the space between the old and new cuticle filled up with waste material (Fig. 7F).

Taken together, the results obtained with these two rescuing constructs (pKK49 and pKK25) imply that elt-5/6 activity in seam cells is essential for proper alae formation, suppression of seam fusion and molting. They further suggest that early L1 lethality is attributable either to elt-5/6 activity in AB and MS descendants or to expression in very early seam cells, before pKK49 gives robust expression in seam cells ∼2 hours after their birth. Finally, they reveal an apparently continuous requirement for elt-5/6 in maintaining seam identity and/or repression of their fusion throughout post-embryonic development.


Background

For over 30 years, the vulval development of C. elegans has been an important model in which to study mechanisms underlying the development of complex organisms [1, 2]. However, most studies of vulval development focused on cell fate specification and inductive interaction during the third larval (L3) stage [3]. The subsequent development of induced vulval cells, during which cell fates determined in the L3 stage are "executed" has been less studied. During the fourth larval (L4) stage, these cells undergo a complex series of morphogenetic events accompanied by dynamic changes in gene expression patterns. This makes it a potentially powerful system in which to study gene regulation during terminal differentiation and mechanisms that underlie complex morphogenetic processes [4–10].

Recent studies of vulval development during the L4 stage follow the detailed description published in 1999 [7]. This work, based on electron microscopy of serial sections and fluorescent labeling of cell-cell junctions, revealed the general sequence of events during the L4 stage. First, vulval cells migrate from where they were generated (near the original positions of vulval precursor cells induced in the L3 stage) toward the center of the future vulva. Second, these cells extend processes and fuse with one another such that they form a dorsal/ventral stack of seven toroids, called vulF, vulE, vulD, vulC, vulB2, vulB1 and vulA. Most of these toroids are syncytial cells with two or four nuclei. The only exceptions are vulB1 and vulB2, which remain unfused, but nevertheless arrange themselves in a ring configuration. Subsequently, the shapes of these cells change further, forming the adult structure that serves as the conduit for developing embryos and for sperm when mating with a male. During this process, additional cell-cell connections are made vulC and vulD make connections to the vulval muscle cells that open the vulva during egg laying, vulE makes a structural connection to lateral hypodermal cells and vulF makes a connection with uv1 cells of the uterus.

Additional studies led to identification of a number of genes involved in this stage of vulval development and understanding of some morphogenetic processes. Polarized migration of vulval cells requires the signaling protein SMP-1/semaphorin and its receptor PLX-1/plexin, as well as small GTPases MIG-2 and CED-10 (members of the Rho/Rac family) and the GTP/GDP exchange factor UNC-73/Trio [8]. Some of these proteins show polarized localization in each vulval cell. Fusion of vulval cells into syncytial toroids requires fusogens AFF-1 and EFF-1 [11, 12]. The zinc finger transcription factor VAB-23 is a target of regulation by the EGF pathway during the L3 stage, and regulates expression of genes including smp-1, thereby linking vulval induction to regulation of morphogenesis in the L4 stage [13]. Finally, morphogenetic movements that shape the developing vulva are a result of complex interplay of various forces operating among the vulval toroids [9]. These forces include contraction of ventral toroids, requiring contraction of actin microfilaments and regulated by the Rho kinase LET-502 [9], as well as generation of dorsal lumen through transient invasion of the anchor cell into the developing vulva [10].

A separate line of investigation looked at genes that are differently expressed in the seven cell types and mutations that affect their expression. Approximately 30 genes are now known to exhibit cell type specific expression among vulval cells in the L4 and/or the adult stage ([14] and references therein). Importantly, each cell type expresses a unique combination of genes, while each gene may be expressed in a single vulval cell type or in multiple cell types. Moreover, the timing of gene expression shows considerable complexity. Expression of different genes in a single cell type can initiate at different time points, and expression of a single gene in different vulval cell types can start at different time points.

The progress in understanding how expression of these genes is controlled has been slow, probably because it is relatively difficult to isolate mutations that affect cell fate or gene expression during the L4 stage. This may be because many of the genes involved in this stage of vulval development are pleiotropic and are required for earlier stages of development [15, 16]. Among the classical lineage mutants studied by Horvitz et al., only lin-11 appears to have a phenotype consistent with cell fate change at this stage [17, 1]. Additional genes (e.g. lin-29, egl-38, cog-1, bed-3, nhr-67, vab-23) were isolated from other screens [18–22, 13]. However, many more genes are likely to regulate this stage of vulval development given the complexity of this system.

Among the known genes regulating gene expression in the L4 stage vulva, a subset demonstrates a possible connection to the heterochronic pathway regulating stage-specific gene expression. In particular, lin-29 (encoding a zinc-finger transcription factor) is a well-known heterochronic gene regulating the L4-to-adult transition [23]. Moreover, bed-3 (encoding a BED-type zinc-finger transcription factor) was recently discovered to be regulated by blmp-1, another component of the heterochronic pathway [24] (our results not shown). These results suggest that the timing mechanism operating throughout the entire body of the worm feeds into vulval development at specific time points, allowing for precise temporal control of gene expression. However, details of how the temporal sequence of events is regulated within the L4 stage, and how the heterochronic pathway regulates this sequence, are unclear.

In relation to these possibilities, one limitation of previous analyses of L4 development has been the lack of precise timing information. In various contexts, L4 stage animals were classified as "early", "middle" and "late" without a precise definition of each phase (e.g. [4, 5]). In order to fully understand vulval development in the L4 stage, further studies must rely on improved description of developmental timing. Here, we present a further subdivision of the L4 stage into sub-stages (L4.0 to L4.9) based on morphological criteria in the vulva as observed by Nomarski differential interference contrast microscopy. This scheme allows staging of an L4 animal at approximately one hour resolution without the need to follow an individual animal over the course of its development. We correlate our sub-stage scheme with developmental timing when the worms are grown at 20 °C. We also present improved measurement of gene expression timing for several well-characterized vulva-expressed genes.


Molecular Genetics of C. elegans Development

My lab is interested in the process of morphogenesis, the development of shape and form. What are the molecules that regulate the behaviour of cells as they change their shape, position and adhesiveness to generate their three-dimensional form during morphogenesis? The answer to this question will certainly add to our understanding of the role of cell adhesion and cell signaling during tissue inflammation and metastasis. We use the genetic model organism Caenorhabditis elegans to study simple examples of morphogenetic movements. The well defined anatomy of C. elegans will allow us to analyze these processes at a level of precision not easily attainable in other organisms. My lab uses genetic, molecular biology, biochemistry, and state of the art video microscopy techniques to elucidate the mechanisms by which tissues and organs are generated. In previous work we have shown that ephrin signaling is required for proper C. elegans morphogenesis.

Ephrin/Eph Receptor Tyrosine Kinase Signal Transduction in C. elegans Morphogenesis:

The Eph receptor tyrosine kinases and their ligands, the ephrins, are an exciting class of molecules that play a wide variety of roles in development, including axon guidance, blood vessel formation and cancer. C. elegans mutants that are defective in ephrin signaling have abnormal morphogenetic cell movements during embryogenesis and as a result usually die . The identification of ephrins and their receptor in an animal amenable to genetics makes it feasible to dissect the entire network of ephrin signaling in an organism. To understand the roles of Eph signaling during C. elegans development we will identify the downstream genes of the Eph receptor and how other genes might act redundantly or in parallel with Eph signaling during morphogenesis. These studies complement the approaches taken to understand Eph signaling in more complex animals and will expedite our understanding in the signal transduction pathways controlling morphogenesis. Further, Eph signaling has been linked to events of vertebrate neurogenesis, angiogenesis, and cancer, the latter of which is a prime candidate for anti-tumour therapies. It is expected that work done in model organisms will generate mechanistic information required to improve the efficacy of such treatments.

C. elegans and Cancer Research

Ye have made your way from the worm to man, and much within you is still worm” Freidrich Nietzsche (1844-1900)

Eph RTKs, Cancer and My Fascination with Development:

I hope to convince you why our research is indeed an important aspect of cancer research. Keep in mind that cancer research is a multifaceted discipline ranging from clinical trials, drug development, epidemiology, and basic research. The latter is the heart of our continued efforts in cancer research. Specifically I will address the following 3 questions:

  1. How is the study of cell movements related to cancer and why we use the model organism C. elegans?
  2. What genes we study and their role in cancer? Specifically Eph RTKs, a gene called vab-1 in C. elegans.
  3. How can we use genetic approaches to identify components in this signaling pathway?
Why use C. elegans?

Animal development is an incredible feat of biological regulation. How are cells, tissues, and organs governed to reach their proper size, shape, and pattern? You can think about cancer as a developmental process, but a developmental process gone wrong. The philosophy of our research is that if we are going to fix something we have a better chance at fixing if we to know how it works in the first place. The Chin-Sang lab is interested in studying a cell and tissue behaviour called morphogenesis. That is, how cells change their shape and size and move during development. So what has all of this got to do with cancer? As I mentioned earlier cancer is a developmental process and currently there is a huge field of cancer research that is devoted to understanding how cells divide and multiply. What genes are turned on or off to tell the cell when to divide or when to stop dividing? We know that tumours arise because of unregulated or too much cell division. Correct? Yes, but this is only one aspect of cancer- in fact we can have benign tumours. What usually makes cancer life threatening is when a tumorous cell invades surrounding tissues or breaks away and travels to distant parts of the body to form yet another tumour- a process called metastasis. Well, surprisingly these cell movements during metastasis are very similar to cell movements seen in the developing embryos of all animals. So if we are going to understand metastasis we need to understand how cells naturally move.

We use this microscopic worm called Caenorhabditis elegans to study some very simple aspects of cell movements. C. elegans are primitive organism that share many biological characteristics of humans. What scientists have learned in the past 20 or so years is that very basic cellular machinery that control mechanisms such as cell division, movement and growth is highly conserved throughout evolution. This means that if you study single cell yeasts, worms, flies or a mouse, many of the genes that control cell growth and development in these organisms have direct counterparts in humans and therefore it has much significance to understanding basic cell biology in humans. The importance of the use of simple model organisms for human diseases is exemplified by the 2002 Nobel Prize in Physiology or Medicine which went to three of C. elegans researchers (Note: I should mention that it also went to yeast researchers in 2001 and fly researchers in 1995). In 2002 the Nobel prize was awarded to Sydney Brenner, Robert Horvitz and John Sulston who used C. elegans to give us significant understanding on genes that regulate program cell death or apoptosis—which has significant relevance to cancer as tumours cells seem to have lost their ability to respond to programmed cell death. The Chin-Sang lab’s research objective is to use very similar genetic approaches as these Nobel laureates to understand the molecular mechanisms that control cell movements.

How do we do this?

We go about this by isolating and characterizing mutants that have defects in cell movements early in the developing embryo. You can imagine that cells must be positioned properly in the embryo to function properly. Cells such as neurons, muscles, gut, and epidermis must find their normal position in the embryo, as such if these movements are all messed up it usually leads to embryonic lethality. We cloned the genes responsible for some of these early cell movements and they encode an Eph Receptor Tyrosine Kinase and the ligands for this receptor. In Figure 1, the VAB-1 Receptor is shown in Blue and one of its ligands shown in Green are expressed on neuroblast the future nervous system. Mutations in either the receptor or ligand lead to abnormal neuroblasts movements during embryogenesis and as a result the embryo usually dies.

What are Eph Receptors and what do they have to do with Cancer?

Eph RTK in cancer regulation:

These molecules are found on the cell’s surface and belong to a large family called the Receptor Tyrosine Kinases or RTKs. The RTKs are a well-established molecule in oncogenesis as overexpression of certain RTKs can lead to tumour formation. Hence, many of these RTKs and their ligands serve as cancer drug targets (e.g.Trastuzumab/ Herceptin ®, imatinib mesylate/ Gleevec® , gefitinib/ Iressa™ , and Avastin™.) The class that we are particularly interested in are the Eph RTKs. They are the largest family of vertebrate RTK- you and I have 14 different Eph receptors in our body. This large number has in part complicated our understanding of how these RTKs signal. However, in C. elegans there is only one receptor making it easier to elucidate the entire pathway in an organism.

So what do we know about Eph RTKs and Cancer?

There is considerable evidence for the roles for Eph RTKs in both metastasis and tumour formation. These receptors and ligands are frequently overexpressed in a wide variety of cancers. For example EphA2 is overexpressed in 40% of all Breast Cancers. I should stress the role of Eph receptors in cancer regulation is not that clear. The downstream signaling mechanisms are different from most other RTKs. But these RTKs have sparked a great deal of interest in cancer researcher because they have been shown to regulate many types of cancers. As such, the Eph RTKs, their ligands, and effectors represent targets for cancer drug development. However, before drug development proceeds we need to fill in significant gaps in our understanding of how these receptors signal. What molecules do the cells use to communicate with each other? Recently we showed that the VAB-1 Eph RTK inhibits the worm version of the human tumour suppressor gene called PTEN.

The Research strategy:

Classical Genetic Analysis:

C. elegans has become a choice genetic model organism for many researchers. Because of its fast life cycle, and small cell number we can genetically manipulate these organisms and observe the consequences at a single cell resolution. Various “genetics tricks” can be used to identify the genes that encode the molecules that enable cells to communicate with each other during development. I only mention 2 below and focus on the logic rather than the details:

Further reading on interpreting genetic modifiers can be found in these classic papers by Leonard Guarente and Avery and Wasserman. Also see this wormbook chapter.

The first approach is called Synthetic lethal Screens:

The logic is as follows: Say you have two genes A and B. A mutation in A does not lead to a phenotype. In fact it is believed that most genes when mutated do not lead to a phenotype. If you have a mutation in gene B you may not get a phenotype for the same reason. But when you make a double combination, that is, two mutations A and B you get a phenotype- in this case death. This is what geneticists call synthetic lethality and it tells you genes and A and B have functional relationship. The easiest way to look at this is that gene A and gene B function in a parallel or redundant pathway. That is, bothA and B are required for some essential developmental function “X”. Knock out A by mutation and it is still ok because B is present, knock out B and it is still ok because A is present. But the double combination leads to a phenotype –lethality.

There is another genetic method called suppressor analysis, which is essentially the opposite of synthetic lethality or enhancement. That is, you start with a phenotype and you identify mutations in other genes that restore the phenotype back to wild type or normal.

Suppose you have a regulatory pathway (See Figure 3): A turns off B , B turns off C and C turns on or Activates X. “X” is an arbitrary developmental process. When A is on this leaves B off, which in turn lets C come on therefore X is ON. Therefore the net effect of A is to turn on X. If there was a loss of function mutation in A such that A is off this allows B to be on which in turn turns off C which can’t activate X leading to a phenotype. If the only function of gene A is to turn off gene B then a suppressor of mutant A would include loss of function mutations in B as this mutation bypasses the need for gene A. Other potential suppressors include gain of function activating mutations in C or X. This type of suppressor approach is what geneticists call suppression by epistasis.

Biochemical Approaches and Yeast 2 Hybrid Screens:

Although researchers like to use phrases like “the awesome power of genetics” keep in mind that genetic approaches are powerful, however they are not all powerful. We are also using molecular and biochemical approaches such as affinity chromatography and yeast 2 hybrid screens to identify components that physically associate with the VAB-1 Eph RTK.

This combined approach of genetics and biochemistry is an effective strategy in deciphering molecular pathways controlling development. Using genetic approaches we have isolated suppressors and synthetic lethal genes that may work with ephrin signaling in C. elegans. From our biochemical work we have identified two new proteins that associate with the Tyrosine Kinase of VAB-1. Both genes have been shown to regulate cancers in humans. Taken the data as a whole, that Eph RTKs are involved in cancer regulation and that we had identified physical interactions and genetic interactions with know tumour suppressor genes as well as genes involved in promoting cancer we believe that we are on our way to understanding how the Eph RTK signal transduction mechanism regulates cancer in humans.

Reverse Genetics: We live in a time when the entire DNA sequence of many organisms is known- The era of Genomics and Proteomics. This information has shaped the way researchers think about understanding how genes and their products (proteins) function at the cellular level and at the organism level. Yes, we too are interested in the growing field of reverse genetics and the Chin-Sang lab has planned many experiments using this technology.