INDUCTION OF THE PHARYNX

Pharynx of the adult worm:

- is a tube in three sections

1. Corpus – joins the buccal cavity to pump food further in
2. Isthmus – moves food by peristalsis
3. Terminal bulb – grinds food finely to prepare it for the intestine
- is a highly muscular organ
- contains other cell types such as nerves and glands

Embryonic Development:

- arises from descendants of ABa and MS blastomeres

- Part of the pharynx arising from ABa:
o requires 2 successive inductive signals to form
1. A repressive signal coming from the P2 cell
 normally prevents formation of pharynx from ABp
 if ABa and ABp are interchanged, ABp will form the anterior pharynx instead of
ABa (meaning cell position plays a greater role than cell lineage)
 if P2 is prevented from touching ABp, then ABp will also form the pharynx
(meaning P2 normally signals ABp to suppress pharynx formation)
 repressive signal is encoded by apx-1, a maternal effect gene (anterior pharynx
excess
 Embryos that lack APX-1 show formation of anterior pharynx from both
ABa and ABp blastomeres
 apx-1 codes for a Delta-like ligand
 the receptor is encoded by another maternal effect gene, glp-1 (effect on germ
line proliferation), whose product is a Notch-like receptor
 Embryos that lack GLP-1 also have ABp developing as ABa
 unlike apx-1-, glp-1- mutants do not go on to form the pharynx from the
two AB blastomeres (because ABa’s default is not to form the pharynx )
o depends on positive inductive interaction
o isolated AB cells do not produce any pharynx
2. A positive signal coming from the descendants of MS
 laser ablation of the MS cell between 8- and 12- cell stage prevents ABa from
forming the pharynx
 at the 12-cell stage…
 MS blastomere touches two ABa granddaughter cells (ABalp and ABara)
 MS blastomere releases the second signal responsible for inducing the
pharynx
 In apx-1- embryos, the descendants of the ABp cell that produce the pharynx are
ABprp and ABplp which contrast MS at the 12-cell stage
 Receptor for this signal is also GLP-1 (because mutant embryos do not form a
pharynx and in other regards, the two lineages behave the same)
 Glp-1 mRNA is uniformly distributed in 8-cell stage but the GLP-1
protein is only found in AB descendants
  This is due to a differential translation regulated by a sequence in the 3’-
UTR, with translation in the posterior being repressed by a cascade of
factors that are ultimately controlled by PAR-1
 GLP-1 disappears in the 28-cell stage (where there are 16 AB
descendants)
 Ligand for GLP-1 is expressed by MS and is distinct from the ligand
expressed from APX-1
 There is a double requirement for GLP-1
1. As the receptor mediating repression of pharynx formation by the
action of P2 on ABp
o If the nonpermissive temperature is given at around the four-
cell stage, then the phenotype is just like the maternal effect
phenotype of axp-1-, with ABp as well as ABa forming the
pharynx
2. As the receptor mediating positive induction of the pharynx
formation by the action of MS on Aba
o If the nonpermissive temperature is maintained until the 12-cell
stage, then the phenotype expressed is just like the maternal
effect glp-1 null mutant, with equivalent cell divisions of ABp
and ABa but no pharynx formation
- Formation of the pharynx is dependent on the zygotically expressed gene pha-4, encoding a
winged transcription factor that is homologous to FoxA genes in vertebrate gut development
o Pha-4 is activated by T-box transcription factors which become upregulated in both ABa and
MS derived parts of pharynx
o In ABa derived parts, they are genes tbx-37 and tbx-38.
o In MS-derived parts is the gene tbx-35
 Upregulated by products of zygotic genes med-1 and med-2 which encode
GATA-type transcription factors and are activated by SKN-1
o PHA-4 is responsible for controlling the expression of pharyngeal genes in all the component
cell types of the organ through a “pharyngeal enhancer”
o Among cell-type-specific genes activated are hlh-6 which encodes a bHLH factor controlling
gland differentiation, and tbx-2 which controls muscle differentiation
o The loss-of-function mutant of pha-4 lacks the entire pharynx (both the parts from ABa and
MS)
o There is a requirement of pha-4 throughout development for both early and late
differentiation events, the level of expression increases during development (promoters with a
high affinity for PHA-4 are turned on early and those with lower affinity are turned on later
when the conc of PHA-4 proteins has been built up to a sufficient level.
o PHA-4 also has functions in gonadal development and affects the lifespan of the adult worm

Development of the Vulva

- the vulva is an epidermal structure formed in larval life around the mid-ventral opening of the
gonad that is used for egg laying and mating
- Formation is controlled by Epidermal Growth Factor (EGF)-like signal from an internal cell:
Anchor cell
- Arises from three posterior daughters of the ectodermal cells P5, P6, and P7
o P5p: seven vulval descendants
o P6p: eight vulval descendants
o P7p: seven vulval descendants
- in the fourth larval stage, the 22 vulval descendants undergo various movements and fusions to
form the vulva itself
- Surrounding cells (P3p, P4p, and P8p) are competent to make the vulva, but in normal
development, they divide once to make two cells that later enter the syncytial hypoderm
- Conventions used:
o Formation of 8-cells: primary fate (followed by P6p)
o Formation of 7-cells: secondary fate (followed by P5p and P7p)
o Formation of 2-cells: tertiary fate (followed by P3p, P4p, and P8p)
- Hermaphrodites can reproduce without a vulva (larvae chew their way out of the body)
o mutations are hypomorphic (cause partial loss of function), with null alleles of the same
genes being lethal
o main classes: vulvaless and multivulva
- The six P3p-P8p cells are said to make up an equivalence group (each are all competent to from
vulva and can replace one another)
o shown in relation to the gonadal anchor cell which lies internally adjacent to P6p
o if anchor cell is removed by laser microbeam ablation, then all P3p-P8p cells will follow
tertiary fate. (no vulva produced)
o if one of the P3p-P8p cells is removed by laser microbeam ablation, then one of its
neighbors will take its place. (normal vulva produced)
o if anchor cell is moved relative to P3p-P8p cells, whichever three of the P3p-P8p cells
nearest will form the vulva (normal vulva produced/ in cases of displaced gonad mutants)
- Anchor cell ligand
o homolog of EGF encoded by lin-3
o receptor is an EGF receptor homolog encoded by let-23
o Ras protein homolog encoded by let-60
  Ras is a protein that functions as binary molecular switches that control
intracellular signaling networks
o Loss of function mutants: vulvaless phenotype
o gain of function mutants: multivulva phenotype
o double mutant combinations
 combination of let-23 and let-60 gof gives a multivulva phenotype
 ras requirement lies downstream of the EGF receptor homolog
o It is possible to visualize a gradient of EGF activity centered on P6p in worms that are
transgenic for an EGF reporter gene
o EGF signaling gradient should be enough to generate three cell fates, there is also a
secondary signal emitted by P6p that activates the Notch pathway in P5p and P7p
o Combination of both EGF signaling and P6p signaling serves to control the development of
the vulva.

Distal Tip Cells:

- Somatic cells whose function is to maintain the neighboring germ-cell nuclei in mitosis
- found on the tip of each branch of the gonad
- They are two cells derived from Z1 and Z4 (which are progeny of MS blastomere)
- Specified by the same Wnt-like mechanism that is earlier responsible for the E-MS decision
- Two distal progeny of Z1 and Z4 experience a high ratio of SYS-1 to POP-1 and become distal tip
cells
o SYS-1 has been found to be similar to Beta-catenin which functions in regulation and
coordination of cell to cell adhesion and gene transcription
o POP-1 which functions in a Wnt pathway that controls cell migration and cell polarity
- Proximal daughters of Z1 and Z4 experience low and repressive POP-1 form the anchor cells that
control vulva formation
- As the gonads grow, the germ-cell zone elongate that such cells leave the range of influence of
the distal tip where they stop undergoing mitosis and start undergoing meiosis
- Ablation of distal tip cell causes all remaining mitotic nuclei to enter meiosis
- Early ablation will create all sperm, late ablation will result in a nearly normal arrangement of
oocytes and sperm
- distal tip cells act as expression of lag-2, a homolog of Delta.
- receptor is encoded by glp-1 the same gene required for pharynx induction (present on the
germ-cell syncytial membrane)
- Zygotic loss of function mutations of glp-1 or lag-2 have the same effect as distal tip cell ablation

Programmed cell death

- during normal c. elegans hermaphroditic development,

o 131 cells die out of the 1090 generated,
o mostly during a death wave from 250-450 minutes of development
o mainly small cells and collectively represent only 1% of total biomass
o most deaths are autonomous and occur shortly after the cell was born
o some spend on signals from neighboring cells, shown by the fact that certain cells
normally designed to die survive if their neighbor has been ablated by laser irradiation
- c. elegans cell death is mostly similar to mammalian apoptosis, with the condensation of the
nucleus, a shrinkage of the cell to a membrane bound body, and engulfment by neighboring
cells
- Cell-death-defective (ced) mutants affect all the cell deaths within the organism
o other mutants affect the decisions of some particular cells to die
o most ced mutants interfere with the engulfment of dead cells
o three are components of the actual death program
 loss-of-function mutant of ced-3 and ced-4, all cells normally destined to die
survive
 ced-9, there is loss-of-function and gain of function
 in loss-of-function mutants: there is excessive cell death
 in gain-of-function mutants: there is some survival of cells that normally
die
 overexpression of wildtype ced-9 also can lead to excess cell survival
 double mutants of ced-9-; ced-3- or ced-9-;ced-4- also show target cell survival,
 thus the normal function of ced-9 is to repress ced-3/4 which
themselves are downstream of ced-9 and necessary to execute the
death program
 ced-9 encodes for the homolog of the mammalian protein BCL2
 discovered originally as an oncogene product
 cytoplasmic protein that is an inhibitor of apoptosis
 CED-9 and BCL2 are interchangeable
 worms of transgenic for mammalian BCL2 show inhibition of cell death
 ced-9- mutants can be rescued by BCL2
 ced-3 encodes for a homolog of interleukin 1β-converting enzyme (ICE)
 a cysteine protease that cleaves at Asp-X sequences
 prototype member of the family of capases which are the enzymes tht
actually bring about cell death
 the targets for CED-3 protease include poly-ADPR polymerase (involved
in DNA repair), lamins (nuclear membrane proteins) and other nuclear
proteins
 CED-3 itself will cause apoptosis if introduced into mammalian cells
 ced-4 encodes for a protein whose mammalian homolog is Apaf1
 activates procapase 9
 CED-4 similarly activates CED-3 and is inhibited by CED-9
- Final stage of Apoptosis: engulfment of apoptotic cells by neighboring cells
o c. elegans does not possess specialized phagocytes and the engulfment is carried out by
non-specialized cells
o a critical step is the recognition of a phospholipid, phosphatidyl serine , on the surface of
the cell about to be engulfed
o a number of ced genes are defective in engulfment and their homologs are thought to be
important for the properties of mammalian phagocytes
 ced-1 encodes a homolog of mammalian SREC (scavenger receptor from
endothelial cells)
 ced-7 encodes an ABC (ATP-binding cassatte) trasporter protein, one of a large
class of proteins responsible for transport of small molecules and ions across
cell membranes
- as with asymmetrical cell division, the discovery of programmed cell death is an area which C.
elegans genetics has made an important contribution to general cell biology
o the important contribution is the discovery of a previously unexplored gene program that
promotes cell death (ced)
o studies on c. elegans by Sydney Brenner, H. Robert Horvitz, and John E. Sulston awarded
them the Nobel Prize for Medicine or Physiology of 2002
o applications of their research include the control of cancer development to stimulate
death gene activity in cancer cells and to stimulate ced-9 activity to address
neurodegenerative problems that are characterized by massive cellular death