3.1.         Anterior-posterior axis formation in C.elegans

The position of sperm pronucleus determines the differentiation of the anterior-posterior axis in C.elegans. Cytoplasmic movement initiates by centriole of sperm pronucleus when it enters within the oocyte cytoplasm. The cytoplasmic movement drives the male pronucleus to the nearest end of the oocyte and that end becomes the posterior pole. Sperm and several PAR ("partitioning") proteins responsible for coordinating the integration of morphogenesis, cell division and cell specification.

The sperm supply a protein, CYK-4, which cause activation of GTPases in the egg cytoplasm. GTPases (G proteins) responsible for an alternation of cytoskeletal proteins, thus alternation of cytoskeletal proteins takes place in turn cause activation of the egg actin microfilaments to reposition PAR proteins, which pushing actin microfilaments anteriorly.

If CYK-4 has been knocked down with RNAi or by mutation mutated then as a result embryos lack polarity and die. PAR proteins also cause localization of the P-granules (localization of the P-granules requires microfilaments but can occur in the absence of microtubules) is ribonucleoprotein complexes that specify the germ cells and it is also a collection of translation regulators. The proteins of these P-granules include RNA helicases, polyA polymerases, and translation initiation factors. P-granules move toward the posterior end of the zygote, shortly after fertilization, so that they enter only the blastomere (PI).   

The P-granules of the P1 cell remain in the posterior of the P1 cell and when P1 divides passed to the P2 cell as the same move to P3 and after that P4 and the P4 progeny become the sperm and eggs of the adult. Segregation of P-granules to the posterior of the cell get prevented when zygote treated by cytochalasin D whereas demecolcine (a colchicine-like microtubule inhibitor) fails to stop this movement.

3.2.         Right-left Axis–

During the division of the AB cell differentiation of the dorsal-ventral axis of the nematode is seen. AB cell divides and forms two AB daughter cell, one being posterior called ABp and one AB daughter cell being anterior called ABa. At the 12-cell stage, the left-right axis is specified. The EMS cell divide to form MS cell and the MS cell contacts half the "granddaughters" of the ABa cell. This pattern distinguishing the right side of the body from the left side. Asymmetric signalling is done by Delta in the MS blastomere, which activates Notch in the cell. This signalling makes the right side of the larva diverge from the left.

3.3.         Control of blastomere identity

Both the conditional and autonomous modes of cell specification occur in C. elegans. When the first two blastomeres are experimentally separated both modes can be seen. Autonomous development takes place within PI cells without the presence of AB, cause the generation of all the cells it would normally make and generating the posterior half of an embryo. The isolated AB cell makes only a small fraction of the cell types which it would normally make. AB blastomere has conditional specification; it is proof because isolated AB cell’s drive ABa fails to make the anterior pharyngeal muscles that it would have made in an intact embryo and it needs to interact with the P1 cell’s descendants in order to develop normally.

3.4.         Autonomous Specification

The determination of the PI lineage cell fates determined by internal cytoplasmic factors in spite of interactions with neighbouring cells. The P-granules are morphogenetic determinant, and they act throughout translation regulation. The SKN-1, PAL-1, and PIE-1 protein are transcription factors that act essentially to establish the fates of cells derived from the four Pl-derived somatic founder cells, eg., MS cell, E cell, C cell and D cell.

The fate of the EMS blastomere generates the posterior pharynx and it is controlled by maternally expressed SKN-1 protein. The skn-1 protein has a DNA-binding motif similar to bZip (family of transcription factors). After the first cleavage, posterior blastomere P1 have the capability to produce pharyngeal cells. MS cell produces pharyngeal muscle cells when isolated and only MS cell which gets a drive from EMS has the ability to generate pharyngeal tissue.

Mutant of skn-1 gene causes lack of pharyngeal cells and show that the fate of pharyngeal cell determine by maternal factor and by nature it is autonomous. Lacking both pharyngeal mesoderm and endoderm derivatives of EMS found in homozygous skn-1 -deficient mothers, due to this mutation embryo form an extra hypodermal (skin) and body wall tissue where the intestine and pharynx form in non-mutant.

SKN-1 is a maternal protein, and it activates the transcription of at least two genes and whose products are also transcription factors. (1) med-1:- which specify the entire fate of the EMS cell and expression of med-1 in other cells can cause non-EMS cells to become EMS. (2) med-2.

A second putative transcription factor, PAL-1, is also required for the differentiation of the PI lineage differentiation. The somatic descendants of the P2 blastomere require PAL-1 for their normal development.

Somatic cell types derived from the C and D stem cells which are not present in PAL-1 mutant embryo. MEX-3 protein is an RNA-binding protein inhibit the translation of pal-1 mRNA, in turn, regulate PAL-1. PAL-1 is absent where MEX-3 is expressed. PAL-1 is seen in every MEX-3-deficient blastomere where MEX-3-deficient. PAL-1 expression and its activation in EMS cell inhibited by SKN-1. 

PIE-1 is a third putative transcription factor which is essential for germline fate. PAR-1 protein causes the expression of PIE-1 in P blastomeres and PIE-1cause inhibition of function of both SKN-1 and PAL-1in the P2 and subsequent germ line cells. If germline blastomeres get mutated for maternal Pie-1 gene then it adopts somatic fates, i.e. P2 cell behaving similarly to a wild-type EMS blastomere. Totipotency of the germ cell lineage get preserve by PIE-1 as well PIE-1 cause the repression of the establishment of somatic cell fate.

3.5.         Conditional Specification

The endoderm cell lineage developed through conditional specification also. The P2 blastomere is a sister of EMS and resides as the neighbour of EMS. At the 4-cell stage, P2 gives the signal to EMS through Wnt signalling,  at this place MOM-2 peptide analogous to Wnt and MOM-3 analogous to Wnt receptor protein Frizzled, as a result of EMS divide into an E cell (which produces the intestinal endoderm) and an MS cell (which produces mesodermal muscles). Wnt signalling specific to E call fate.

The EMS cell will divide into two MS cells, When P2 cell is removed at the 4-cell stage of embryo then endoderm is not formed. When P2 blastomere is recombined with EMS then it forms endoderm but when P2 blastomere is recombined with ABa, ABp or both AB derivatives is does not form endoderm. The SKN-1 protein causes activation of gene encoding transcription factor MED-1 and MED-2 as a result beginning of specification of the MS cell takes place.

The POP-1 signal blocks the ability of MED-1 and MED-2 to activate the tbx-35 gene, which activates mesodermal genes (PHA-4 in the pharynx and muscles HLH-1 in muscles) in C.elegans, as a result, inhibit the prospective MS cell to become MS and also block the pathway of E (endoderm). POP-1 get down-regulated by Wnt signalling in EMS daughter cell destined to become the E cell. Both EMS daughter cells become E cells in the pop-1-deficient embryo.

Signals from P2 cell also play a crucial role in distinguishes ABp from its sister ABa. Neurons, hypodermis, and the anterior pharyngeal cells form by ABa and neurons and hypodermal cells form by Abp. Fate of ABa and ABp ( both are univalent cells) are determined by their position within the embryo. Thus if experimentally reverses the positions within embryo then embryo develop normally. EMS blastomere makes contact with both ABa and ABp but P2 cell only make contact with Abp. The GLP-1 protein on the ABp cell makes contact with the APX-1 (anterior pharynx excess) protein (analogous to Delta) on the P2 blastomere. When ABa force to make contact with P2 cell convert into ABp-type cell.

Within the embryo, ABp is transformed into an ABa cell. When the mother of the embryo has mutant glp-1. The GLP-1 protein is a member of the Notch receptor family, which serve as cell membrane receptors in many cell-cell interactions, present on both the ABa and Abp cells.

In C.elegans, Symmetry between ABa and ABp get break by APX-1 signal, APX-1 signal stimulates the GLP-1 protein solely on Abp blastomere of AB descendant, that it only one get touch by APX-1–GLP-1 protein signalling, in turn, P2 cell, initiates the dorsal-ventral axis and it is also responsible for adoption of particular fate by Abp blastomere that makes them different from that of their sister cell ABa.

3.6.         Gastrulation in C.elegans

In C.elegans gastrulation starts very early, just after the generation of the P4 cell in the 24-cell embryo. At this time, migration of E cell’s two daughters (Ea and Ep) takes place into the centre of the embryo from the ventral side. There they form 20 cells gut through division. Prior to the movement of the Ea and Ep cells, there are a very small and transient blastocoel and inward migration of Ea and Ep cells creates a tiny blastopore. After that P4 cell migrate through this blastopore to a position beneath the gut primordium, which is the precursor of the germ cells.

It migrates to a position beneath the gut primordium. The mesodermal cells move into next mesodermal cells move and the inward migration of the descendants of the MS cell occur from the anterior side of the blastopore, and from the posterior side, the C- and D derived muscle precursors enter. Thus gut gets flank on the left and right sides. After 6 hours of fertilization,  pharynx forming AB-derived cells are brought inside, while through epiboly ventral migration of the hypoblast (hypodermal precursor) cells takes place in turn causes closing the blastopore.

Sealing of the two side of hypodermis done by E-cadherin which is present on the tip of the leading cells that assemble at the ventral midline. The cells movement and development into organs takes place during the next 6 hours, and the formation of a worm with 558 somatic cells from searching for a ball-shaped embryo takes place. While these gastrulation movements give a good "approximation" of the final form but for movement of cells into functional arrangements an additional"cell focusing" is also used.  Here,  along the anterior-posterior axis cells of the same fate get sorting out. Apoptosis of an additional 115 takes place. Formation of sexually mature, hermaphroditic adult worm takes place after four moults and it is containing numerous sperm and eggs as well as exactly 959 somatic cells. Adult C.elegans male contains 1031 somatic cells.

3.7.         Differentiation of the C.elegans Pharynx

Pharynx in C.elegans is generated by two sets of cells. One group of pharyngeal precursors dependent on the maternal skn-1 gene and comes from the EMS cell. The second group of pharyngeal precursors comes from the ABa blastomere give the second group of pharyngeal precursors and it is dependent on EMS cell govern GLP-1 signalling, which activates the transcription factor-encoding pha-4 gene and that activates almost all of the pharynx- specific genes. The pha-4 transcription factor resembles the mammalian HNF3p protein.

3.8.         Vulva formation in C.elegans

C.elegans is a hermaphrodites nematode. C.elegans is male during the early development and at that time gonad produce sperm and store it for future use. They develop ovary during the later stage of development. Fertilization takes place inside the vulva when the egg gets roll by the region of sperm storage, After fertilization, fertilized egg get to pass down outside the body of a vulva.  In C.elegans formation of the vulva characterizes a case in which one inductive signal creates a variety of cell types. Vulva formation takes place during the larval stage, vulva form by six cells called the vulval precursor cells (VPCs), which give rise to 22 cells so that a mature vulva contain 22 cell form during postembryonic (larval) development.

During the L1 larval stage, eleven Pn.p cells (P1.p, P2.p, P3.p,..., P11.p) are formed as the posterior daughters of the Pn ventral neuro-ectoblasts.

Just before the end of the first larval stage, Wnt signalling takes place which induces expression of Hox gene lin-39 in six vulval precursor cells (6 Pn.p, from P3.p to P8.p become VPCs). The Lin 39 expression six vulva precursor cells are formed by vulval equivalence group. The lin 39 also maintains the VPCs as polarized epithelial cells. These six VPCs become competent to respond to an inductive signal or developmental signal that produced later during vulval development or become competent to Lin3 signalling.

3.8.1.     Hox genes and VPC competence group

At the establishment of the third larval stage, vulval precursor cells fates are determined by the collective action of the RTK/RAS/MAP kinase (LIN-3 epidermal growth factor (EGF) signalling) and the LIN-12 Notch signalling pathways. WNT signalling takes place via BAR-1 beta-catenin.

Within P3.p to P8.p expression of lin-39 takes place. Two main roles of lin-39, first, inhibits fusion with hypodermis via inhibition of EFF-1 expression.

(prevention of VPCs fusion) and second stimulating cell division, responsiveness to LIN-3. MAB-5 cause inhibition of lin-39 especially occurs in more posterior cells including P7.p and P8.p.

3.8.2.     Roles of LIN-39

Anchor cell, which is present within gonad, responsible for differentiation of hypodermis cell to vulva precursor cells (VPCs). The anchor cell is specified from two somatic gonadal cells during the end of the L2 stage, from two somatic gonadal cells cause specification of anchor cell. The basement membranes get to break down by anchor cell which is secreted by the gonad and the epidermis and maintain the separation of these two tissues. the anchor cell extends the process to the center of the vulF cells a process get extends by anchor cell and forms a hole in the epidermis. Through the vulE cells, anchor cell can pass and locate the vulF cells.

Anchor cell carried out paracrine signalling and induces vulval differentiation by secreting Lin3 that activates the receptor tyrosine kinase LET-23 (EGF receptor).

A concentration gradient gets the form by LIN-3 protein. The highest concentration of LIN-3 protein is received by the primary VPC closest to the anchor cell and generates the central vulval cells. The two VPCs adjacent to it (P5.p and P7.p) receive a lower amount of LIN-3 and become the lateral vulval cells.  VPCs farther away from the anchor cell become hypodermis because it does not receive enough LIN-3 from anchor cell.

When lin3-Let23 interaction on P6.p takes place. The RTK/RAS/MAP kinase signal transduction pathway carried out. specifies the primary (1°) vulval fate, firstly it activate Let60 (Ras) then it activates Lin 45(Raf), then it activates MEK-2 and MAK-1. The activation of protein block eff-1 block cell fusion. 

MAPK pathway target two transcription factors LIN-3l and LIN-1. LIN-3l and LIN-1 both get phosphorylated by MAP kinase MPK-1. Multivulva phenotype resulted from Inactivation of either lin-1or lin-31. The complex of  LIN-1and LIN-31 disrupted by LIN-31 phosphorylation. Vulvaless phenotype resulted from non-phosphorylated protein. lin-31 mutants during the L3 stage display both vulvaless and multivulva phenotypes Inappropriate divisions of Pn.p cells during the L2 stage display by  LIN-31, suggesting also a role in the proper specification of VPCs.

P6.p mediated lateral signalling, in response to induction by AC signal, as a result, LIN-12 Notch signalling ( juxtacrine signalling ) takes place by the release of Lin 12 ligand, which get bind to its neighbours P5.p and P7.p in turn cause inhibits the expression of the 1° fate (a process termed lateral inhibition) firstly and secondly mediates the secondary (2°) fate in P5.p and P7.p. MicroRNA, mir-61get activated by Notch signal, as a result cause repression of gene that would specify central vulval fate.

Tertiary (3°) fate get adopted by the VPCs (P4.p, P3.p and P9.p ) which receive neither inductive nor lateral signals, in turn, they fuse with the epidermis (hyp7) after undergoing one round of cell divisions.

Model of VPC pattern formation. 1°-2° patterning mechanism shown here within three of the six VPCs (P6.p, P7.p and P8.p). Anchor cell release Lin3 and LIN-3 acts in a graded fashion with P6.p receiving more signal than P7.p. LET-23 activation promotes the 1° fate, inhibits LIN-12 protein levels and cause the production of DSL ligands for LIN-12.  2° fate promoted by LIN-12 activation in turn cause inhibition of  response to LET-23 activation

3.8.3.     Mutational effect

VPCs will not form a vulva if the lin-3 gene is mutated and become part of the hypodermis (skin). If the anchor cell is destroyed, the three outer cells, which normally form hypodermis, generate vulval cells instead.

In the case of lateral signalling other VPCs, P7.p becomes 1° in a lin-15 multivulva mutant in the absence of the gonad. The two cells adopt a 1°-2° or 2°-1° pattern at random If P8.p is also present. P7.p

In the case of sequential signalling, mosaic analysis of let-23.  yellow, 3° fate; red, 2° fate; blue, 1° fate. The genotype of VPCs in the key mosaic animal as follows (-/- lacking let-23 activity) (+/+let-23activity present). all VPCs become 3° in the absence of let-23. In mosaic animals which have let-23 in P6.p but lack let-23 in P5.p and P7.p show that the vulva is usually wild-type. This observation indicates for presumptive 2°VPCs, let-23(+) is not required.

The pattern of VPC cell fates direct level of LIN-3. The six VPCs adopt the 3°-3°-2°-1°-2°-3° pattern in each wild-type case. Animals with reduced lin-3 function (lin-3(rf) show lin-3 reduction of function) display that P6.p induced to 1°. A highly penetrant vulvaless phenotype resulted from a further decrease in lin-3 function. Through anchor cell excessive lin-3  (lin-3(+ + +)) results in an expansion of the pattern, means more than one central cell.

In lin-17 mutants, vulva gets the form by  P5.p and P6.p or may be posterior pseudo-vulva get from by P7.p.

Constitutive 23 mutation causes a MUltiVulva (Muv) phenotype. Typically have a single functional vulva in multivulva hermaphrodites and additional ventral protrusions vulval tissue form each pseudovulva. A psuedovulva posterior to the normal vulva resulted from a mutation within the reversed polarity of P7.p 2° lineage.

There are fewer VPC divisions in the cye-1 mutant.

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