Morphologically defined sub-stages of C. elegans vulval development in the fourth larval stage
© Mok et al. 2015
Received: 18 February 2015
Accepted: 3 June 2015
Published: 12 June 2015
During the fourth larval (L4) stage, vulval cells of C. elegans undergo extensive morphogenesis accompanied by changes in gene expression. This phase of vulval development, occurring after the well-studied induction of vulval cells, is not well understood but is potentially a useful context in which to study how a complex temporal sequence of events is regulated during development. However, a system for precisely describing different phases of vulval development in the L4 stage has been lacking.
We defined ten sub-stages of L4 based on morphological criteria as observed using Nomarski microscopy (L4.0 ~ L4.9). Precise timing of each sub-stage at 20 °C was determined. We also re-examined the timing of expression for several gene expression markers, and correlated the sub-stages with the timing of other developmental events in the vulva and the uterus.
This scheme allows the developmental timing of an L4 individual to be determined at approximately one-hour resolution without the need to resort to time course experiments. These well-defined developmental stages will enable more precise description of gene expression and other developmental events.
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 . 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 . 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 . 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 . Finally, morphogenetic movements that shape the developing vulva are a result of complex interplay of various forces operating among the vulval toroids . These forces include contraction of ventral toroids, requiring contraction of actin microfilaments and regulated by the Rho kinase LET-502 , as well as generation of dorsal lumen through transient invasion of the anchor cell into the developing vulva .
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 ( 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 . 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  (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.
Description of L4 sub-stages defined by morphological characteristics of the vulva
Sub-stages of L4
Approximate time from middle of the L3-to-L4 lethargus
vulA, vulB and vulE have divided, but vulC and vulF have not.
vulC and vulF have divided and a narrow lumen has formed.
Lumen has widened and a prominent kink has formed between vulC and vulD.
vulFs have separated and the apex of the lumen is flat and capped by the anchor cell.
utse is visible as a thin layer separating the vulval and uterine lumens. “Fingers” are formed at the sides of the vulva next to vulB1 and vulB2.
Side of the vulval lumen between vulC and vulD forms a smooth curve.
“Fingers” between vulB2 and vulC are pointed ventrally.
vulFs have migrated closer such that the lumen is narrowed in the dorsal section. The approximate cutoff is when the width of the channel is less than the width of the vulD nucleus.
Lumen is partially collapsed.
Lumen is completely collapsed.
Precise timing of reporter gene expression in the L4 stage
Duration of expression for vulval gene expression markers
Duration of GFP expression
L4.2 to L4.9*
C, D, E, F
L4.1 to L4.9*
 Variable expression in L4.1; consistent in later sub-stages
B1, B2, C
L4.1 to L4.9*
 Variable expression in all sub-stages; low level of expression in B2, C from L4.1 to L4.7
L4.3 to L4.9*
 Expression in E variable in L4.3 and L4.4, consistent in later sub-stages; expression in F variable throughout
No expression in L4
 Expressed in adults only
C, D, E, F
No expression in L4
 Expressed in adults only
L4.4 to L4.9*
L4.2 to L4.9*
D, E, F
L4.1 to L4.7
 Only reporter in this study where expression was obviously turned off during L4.
B1, B2, C, D, E
L4.1 to L4.9*
 Variable expression possibly due to loss of extrachromosomal array
Interestingly, expression of cdh-3::cfp, reported to begin in early-L4, and expression of ceh-2::gfp, reported to begin in mid-L4 , actually started at the same sub-stage L4.1. Although differences in assay conditions could account for the discrepancy, this is perhaps more likely due to differences in the definition of "early-L4" and "mid-L4" used in different experiments, highlighting the need for better staging of L4 animals.
Correlation of L4 sub-stages with other developmental events
Correlation of sub-stages with other developmental events in the L4
From L3-to-L4 molt
Cell migration and fusion
All cells complete migration. vulA, vulC and vulD cells fuse.
Uterine lumen has not started to form.
vulF cells fuse.
Uterine lumen starts to form.
Uterine lumen is fully distended.
vulE cells fuse at L4.8 or later
As with vulval development, the uterus undergoes a complex morphogenetic process during the L4 stage . We did not attempt to correlate all known developmental events in the uterus with our sub-stages. Instead, we looked at an easily assayed aspect of uterine development, the generation of the uterine lumen. We found that L4.2 animals had no visible uterine lumen, whereas L4.4 animals had a large uterine lumen that appeared to be fully distended. Some animals in the L4.3 stage had an intermediate sized lumen, and it appears that the uterine lumen is generated and enlarges during this sub-stage.
Utility of morphology-based staging in studies of late vulval development
An alternative to morphology-based staging is to carry out timed experiments. Individuals or populations at a particular developmental time (e.g. exit of the L3-to-L4 molt lethargus) can be selected, and further observations can be carried out at specific subsequent time points. The major advantage of morphology-based staging is that it is much less laborious. Using this system, any given animal in the L4 stage, provided that it has normal vulval development, can be staged with approximately one hour resolution.
The precise control of the temporal sequence of events during development is a problem that is not fully understood. Although the heterochronic pathway is well characterized, the sequence of events within each stage has received less attention. In particular, development of organs like the vulva requires a complex sequence of morphological changes within a single developmental stage, suggesting that there are additional timing cues which regulate specific developmental events at specific sub-stages. Whether these timing cues are organ-specific is unknown, as is the relationship between these sub-stage cues and the heterochronic pathway. L4 sub-stages described in this report should facilitate the analysis of morphogenetic or gene expression events which occur at specific time points within the L4 stage.
Communication and reproducibility of results is enhanced by definitions of terms. WormBase has developed an ontology of nematode life stages that reconciles most of the terms used in the literature (W. Chen and P.W.S, unpublished results). The sub-stages we have defined here have been added to the Life Stage Ontology. This addition will allow researchers to annotate gene expression and other experiments with these stages, thus providing a more accurate depiction of their observations.
C. elegans culture and timing of L4 sub-stages
C. elegans strains were cultured using standard methods on NGM agar plates seeded with OP50 at 20 °C . The N2 Bristol strain was used as the wild-type. To determine the precise timing of each developmental sub-stage, a population of N2 wild type worms were synchronized using a modified version of the egg-laying protocol described by Lionaki and Tavernarakis (2013) . First, gravid hermaphrodites were allowed to lay eggs on seeded NGM plates for one hour and were subsequently removed. Next, the plates were incubated at 20 °C for approximately 40 h and screened for molting L3 animals based on pale appearance, lack of motility and absence of pharyngeal pumping. The molt-stage worms were then transferred to new seeded NGM plates. Finally, these synchronized populations were examined by Nomarski microscopy as described  at specific time points and the sub-stages were determined.
To analyze the gene expression pattern in various L4 sub-stages, semi-synchronized populations of GFP reporter strains were generated by synchronized egg-laying as described above. After 40 to 50 h at 20 °C, L4 animals were picked randomly and analyzed for fluorescence and the sub-stage.
Strains used are: NH2466 ayIs4[egl-17::gfp]; PS3504 syIs54[ceh-2::gfp]; PS3475 syIs51[cdh-3::gfp]; JU486 mfIs4[egl-17::yfp; daf-6::cfp]; PS3664 syIs65[B0034.1::pes-10::gfp]; PS3527 syIs61[F48B8.6::gfp]; MH1564 kuIs36[egl-26::gfp]; OP171 wgIs171[egl-38::TY1::EGFP::3xFLAG]; BC15833 sEx15833[best-13::gfp]; ZF1638 qwEx197[pax-2::yfp]; ZF1639 qwEx198[pax-2::yfp] [4, 31–33].
Of the transgenic strains we examined, two (PS3664 syIs65[B0034.1::pes-10::gfp] and PS3527 syIs61[F48B8.6::gfp]) were found to be expressed only in the adult and not in L4, as reported previously . The pax-2::yfp transgenes were generated in this study by fusing 3.1 kb sequence upstream of the pax-2 gene to the yfp (yellow fluorescent protein) coding region. Although this fragment is known to contain an enhancer element active in vulval cells, our reporter failed to express YFP in the vulval tissue for unknown reasons.
To assay for cell fusion, we crossed NW1615 plx-1(ev724) jcIs1[ajm-1::gfp]; him-5(e1490) or NW1072 smp-1(ev715); jcIs1[ajm-1::gfp]; him-5(e1490) hermaphrodites with EG7959 unc-119(ed3); him-5(e1490) oxTi405 [eft-3p::TdTomato::H2B::unc-54 3'UTR + Cbr-unc-119(+)] males and F1 progeny carrying oxTi405 were examined. Confocal images were obtained using Zeiss LSM700 confocal microscope. Both confocal and conventional fluorescence microscopy were used to determine whether the cells were fused.
We thank Goh Kah Yee for preliminary analysis of best-13::gfp expression. We thank Marie-Anne Felix for the daf-6::cfp plasmid and strain. Some strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by the Singapore Ministry of Education Academic Research Fund Tier 1 grant (T13-1001-P02). PWS is an Investigator of the Howard Hughes Medical Institute.
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