REPRODUCTIVE SYSTEM - (Part IVa) The egg-laying apparatus

General description - The uterus - The vulva - List of cells - Back to Contents

General description

The egg-laying apparatus consists of the uterus, the uterine muscles, the vulva, the vulval muscles and a local neuropil formed by the egg-laying neurons (EggFIG1). After fertilization embryos pass from the spermatheca to the uterus, an epithelial egg chamber that links the two arms of the gonad. There, eggs develop to the 100 cells stage approximately (roughly 2.5hrs post-fertilization at 20°C) before being expelled into the environment via the vulva, a passage way from uterus to the ventral exterior. Under optimal conditions an adult hermaphrodite will lay 4 to 10 eggs/hour. Egg laying is facilitated by contraction of the vulval muscles, which attach to the lips of the vulva, and possibly the uterine muscles, which encircle the uterus (described in Part IVb). Muscle activity is regulated by motor neurons, in particular motor neurons VCn (VC1-6) and HSNL/R (Part IVb), which synapse on to each other and on to vulval muscle arms forming a neuropil near the vulva. Tissues comprising the egg-laying apparatus arise from several different lineages (see Part I, ReprodTABLE1). As described below, the developing gonad (particularly the gonad AC - Anchor Cell) and the vulva act as organizing centers, recruiting cells from other regions to the mid-body, coordinating cell patterning between different tissues and directing axon guidance and synaptic patterning of the neurons.

The uterus

The uterus consists of an anterior and a posterior lobe joined to a central chamber. The central chamber is joined ventrally to the vulval epithelial tube (EggFIG2A, 2B, 3A). The entire outer (basal) surface of the uterus is covered by a thin basal lamina, the ubl (uterine basal lamina) (EggFIG3C, 3D). The organization of cells that comprise the uterus is most readily apparent during the late L4 stage, after uterus and vulval morphogenesis have taken place, but prior to the onset of ovulation, when the uterus becomes distorted and crowded with embryos. The anterior and posterior uterus lobes are each composed of four uterine toroid epithelial syncytia, ut1-ut4 (EggFIG2A, 2B, 3D; EggTABLE1). ut1-ut4 are joined to each other and to their neighbors both by adherens junctions (aj) (EggFIG2C) and by pleated septate junctions, most robust between ut4 and ut3 and less obvious at the other borders. Cytoskeletal elements are sometimes evident in ut toiroids, running circumferentially, suggesting that the toroids may have myoepithelial properties (Newman et al., 1996 - pdf available from WA Home Page Literature section).


EggTable 1

Cell Description
Syncytial
Nuclei
SPh Precursor cell
ut1-3 toroidal, form uterus arms
yes
4
VU, DU
ut4 toroidal, form uterus arms
yes
6
VU, DU
utse "H"-shaped, attaches uterus to seam
yes
9
AC plus 8 from VU
uv1 ventral, interfacial cell between uterus and vulva
no
1
VU
uv2-3 ventral, interfacial cells between uterus and vulva
no
1
DU
du central dorsal cap
yes
4
DU

SPh, Somatic gonadal Primordium of the hermaphrodite, formed at the L2/L3 molt - see GonFIG1, Reproductive system-Part I. From Newman and Sternberg,1996

The central chamber of the uterus is capped dorsally by the du (dorsal uterine) syncytium and ventrally by the utse (uterine seam) syncytium and uv (uterine ventral) cells, uv1-3 (EggFIG2A, 2B, 2D, 3A-C; EggTABLE1). uv1-uv3 form a multilayered set of flaps, binding the ventral uterus to the most dorsal ring of the vulva, vulF. The utse has a distinctive "H"-shaped structure. The two sides of the "H" attach to the lateral seam of the animal and hold the uterus in place. At the join, the basal lamina is thickened and contains hemicentin (EggFIG3C; Vogel and Hedgecock, 2001). The central portion of utse (the cross bar of the "H") initially forms a hymen membrane between uterine and vulval lumens. Passage of the first egg breaks this membrane and the two lumens become continuous.

Prior to the first fertilization event the uterus lumen is narrow and blocked by a series of inwardly projecting fingers that extend from the uterine lumen wall (EggFIG3D, 5C). After passage of the first egg, the mature uterus retains a few inward septa that may derive from these earlier fingers. In both the developing and mature uterus a continuous thickening or electron dense layer (possibly a glycocalyx or surface coat) is also apparent on the lumenal (apical) membrane, lining projecting fingers and septa(EggFIG9C).

Cells of the uterus arise from DU (Dorsal Uterine) and VU (Ventral Uterine) blast cells of the larval SPh (Somatic gonadal Primordium of the hermaphrodite - Part II-Somatic Gonad; see Lineage Tree; Kimble and Hirsh, 1979; Newman et al., 1996; EggTABLE1). In late L2 one of two somatic gonadal cells, Z1.ppp or Z4.aaa, is specified to become the AC, while the other becomes 1 of 3 VU blast cells (EggFIG4A; Kimble, 1981; Greenwald et al., 1983; Seydoux and Greenwald, 1989; Greenwald, 1997; Karp and Greenwald, 2003).

In late L3 the AC induces VU granddaughters to adopt the "p " fate (EggFIG4B, 5A). p daughters subsequently differentiate into the uv1 and utse cells of the ventral uterus, which lie immediately dorsal of the developing vulva (EggFIG4C, 4D, 5B, 5D; Newman et al., 1995, 1996; Chang et al., 1999). The differentiation of these and many other terminal uterine cells involves dramatic changes in shape and/or fusion to achieve their final morphology (Newman et al., 1996). As described below, the AC also patterns cells of the vulva. This dual induction of vulval and uterine cell fates by the AC ensures that cells forming the physical connection between the uterus and vulva (utse, uv1 and vulF) develop in physical register. The AC also contributes to formation of this connection by creating an opening at the apex of the vulva (see below).

The vulva

The vulva (EggFIG6) is formed from a stack of 7 non-equivalent epithelial toroids or rings (in ventral to dorsal order): vulA, vulB1, vulB2, vulC, vulD, vulE and vulF (EggFIG3B). Each ring is either a single tetranucleate syncytium or two binucleate half-ring syncytia (vulB1 and vulB2). The vulval lumen is lined with cuticle (EggFIG7B). As described below the toroids are formed by vulval cells of two fates: 1° fate (vulE and F) or 2° fate (vulA to D). These cells and the toroids they form express distinct combinations of genes (Inoue et al., 2002) and, potentially, different characteristics and properties. For example, during copulation males locate the vulva with their hook and post-cloacal sensilla (shown in Male Handbook-INTRODUCTION, Part I), possibly in response to signals or characteristics associated with toroids formed by 2°-fated cells (Barr et al., 1997).

Adjacent toroids are joined to their neighbors via adherens junctions (aj) (EggFIG7A-C). Dorsal-most toroid, vulF, is also linked by adherens junctions to the uterine transitional epithelial cells uv1 and uv2, and possibly, the utse (EggFIG3A). Ventral-most toroid, vulA, is linked via adherens junctions to the ventral hypodermal ridge. The vulE toriod stretches laterally and is linked on its basal (outer) surface to the body wall at the lateral seam by a specialized thickened basal lamina (EggFIG3C).

Vulval development spans roughly the same period as uterus development, L3 to late L4. Establishment of the vulva requires the local deformation of existing ventral structures such as the VNC (EggFIG1) and ventral body wall muscles (EggFIG3B), which are deflected laterally in this region to accommodate the vulva. Vulval development can be divided into two phases (i) vulval cell patterning and generation (EggFIG8,9) and (ii) vulval morphogenesis (EggFIG10). The molecular and genetic mechanisms underlying these processes, particularly cell patterning, have been studied extensively and are described in the following reviews and papers and references therein: Greenwald, 1997; Kim, 1997; Levitan and Greenwald, 1998; Eisenmann et al., 1998; Hanna-Rose and Han, 2001; Shemer and Podbilewicz, 2003; Sundaram, 2004; Ceol and Horvitz, 2004).

(i) Vulval cell patterning. The cells that form the vulval toroids are the progeny of ventral hypodermal Pn.p cells (Sulston and Horvitz, 1977; see Lineage Tree). Twelve Pn.p cells are born mid L1. The 6 central cells, P3.p to P8.p, are endowed with equal potential to produce vulval cell lineages and are referred to as VPCs (Vulval Precursor Cells) (EggFIG8A). In L3 the VPCs are patterned so that vulval potential is restricted to the central three cells, P5.p to P7.p. This patterning of the VPCs involves the combined action of 3 inter-cellular signaling events: an inductive signal emanating from the AC (LIN-3/LET-23 MAPKinase pathway activation), lateral signaling between VPCs (LIN-12/Notch) and signals from hyp 7 (reviewed in Greenwald, 1997; Sundaram, 2004).

As a consequence of patterning P6.p expresses a 1° vulval cell fate and P5.p and P7.p 2° vulval fates. The remaining VPC express a non-vulval 3° fate and their progeny fuse with the hypodermis (Sternberg and Horvitz, 1986). 1°, 2° and 3° fate is recognized by the lineage pattern generated by a VPC (EggFIG8B; EggFIG9).

(ii) Vulval morphogenesis. During the final round of vulval cell divisions, the 1° descendants and some 2° descendants detach from the cuticle, allowing the vulval sheet to bend inward and the cells within it to rearrange their cell-cell contacts (EggFIG9D). This invagination step establishes the beginnings of the vulval lumen which continues to expand during morphogenesis. Proteoglycans and their associated glycosaminoglycans, likely expressed in vulval cells, are necessary for this step although their precise role is not known (Herman and Horvitz, 1999; Bulik et al., 2000; Hwang et al., 2003). As morphogenesis continues cells migrate towards the center of the developing vulval primordium and wrap around to meet their anterior/posterior homologs on the other side (EggFIG10; Sharma-Kishore et al., 1999). Homotypic cell fusions occur between cells of homologous fate resulting in the formation of toroid or half-toroid rings (see Inoue et al., 2002 for a useful guide to vulval cell nuclei positions during and after morphogenesis).

As part of the process of joining vulval and uterine lumens, the AC creates a hole in the apex of the developing vulva (EggFIG9A-D). In L3, while Pn.p cells are dividing, the ventral hypodermal basal lamina (bl) and gonadal basal lamina (gbl) breakdown precisely at the site of contact with the AC. The basolateral portion of the AC crosses through this gap, attaches to, then inserts between the descendents of the 1°-fated P6.p lineage cells. This invasion is stimulated by a diffusible signal from the 1° cells (Sherwood and Sternberg, 2003). Later, P6.p terminal progeny fuse together forming a toroid (vulF) around the invading AC process. The AC is then removed by heterotypic fusion with the utse leaving a channel in the apex of the vulva (Newman et al., 1996). When the utse membrane is ruptured, by passage of the first egg, uterine and vulval lumens become continuous.

During late L4 the vulval muscles attach to the vulval epithelial tube and to the body wall (Part IVb). The tube then partially everts (turns inside out) generating the adult vulva in which the lumen is closed until vulval muscles contract (EggFIG11; Sulston and Horvitz, 1977; Sharma-Kishore et al., 1999).

List of cells

(under construction)

1. Late L2/early L3 stage SPh cells that give rise to the uterus

DU Z1.pap (Dorsal Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

 

VUs and AC are of either the 5R or 5L configuration:

5R configuration

VU, Z1.ppa (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

AC, Z1.ppp (Anchor Cell)

VU, Z4.aaa (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

VU, Z4.aap (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

5L configuration

VU, Z1.ppa (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

VU, Z1.ppp (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

AC, Z4.aaa (Anchor Cell)

VU, Z4.aap (Ventral Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

 

DU, Z4.apa (Dorsal Uterine precursor; generates uterus, spermatheca and spermatheca-uterine valve cells)

 

2. L3 stage intermediate blast cells of the uterus

 

The Dorsal Eight (DE) cells (great-grand progeny of the DUs)

DE1, Z1.papaaa (generates anterior arm sp cells)

DE2, Z1.papaap (generates anterior arm sp and sujc valve cells)

DE3, Z1.papapa (generates anterior arm sujn valve cells and ut2-4 uterus cells)

DE4, Z1.papapp (generates uv3, uv2, ut1, du)

DE5, Z1.pappaa (generates du, ut1, uv2, uv3)

DE6, Z1.pappap (generates ut2-4 and posterior arm sujn valve)

DE7, Z1.papppa (generates posterior arm sujc valve and sp)

DE8, Z1.papppp (generates posterior arm sp)

 

DE1, Z4.apaaaa (generates anterior arm sp)

DE2, Z4.apaaap (generates anterior arm sujc valve and sp) WB says male gon sv group

DE3, Z4.apaapa (generates anterior arm sujn valve cells and ut2-4 uterus cells)

DE4, Z4.apaapp (generates uv3, uv2, ut1, du)

DE5, Z4.apapaa (generates du, ut1, uv2, uv3)

DE6, Z4.apapap (generates ut2-4 and posterior arm sujn valve)

DE7, Z4.apappa (generates posterior arm sujc valve and sp)

DE8, Z4.apappp (generates posterior arm sp)

 

p cells (progeny of the VUs)

 

 

r cells (grand-progeny of the VUs)

 

 

The Ventral Twelve (VT) cells (great-grand progeny of the VUs, daughters of p and r cells)

 

3. Adult uterus

ut1

ut2

ut3

ut4

du

utse

uv1

uv2

uv3

ut1

ut2

ut3

ut4

 

4. L3 stage Vulva Precursor Cells (VPCs)

P3.p

P4.p

P5.p

P6.p

P7.p

P8.p

5. Adult vulva


vulA

vulB1

vulB2

vulC

vulD

vulE

vulF


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