sem-4/spalt and egl-17/FGF have a conserved role in sex myoblast specification and migration in P. pacificus and C. elegans

Evolutionary comparisons between Caenorhabditis elegans and the satellite organism Pristionchus pacificus revealed major differences in the regulation of nematode vulva development. For example, Wnt signaling is part of a negative signaling system that prevents vulva formation in P. pacificus, whereas it plays a positive role in C. elegans. We wondered if the genetic control of the second major part of the nematode egg-laying system, the sex muscles, has diverged similarly between P. pacificus and C. elegans. The sex muscles derive from the mesoblast M, which has an identical lineage in both species. Here, we describe a large-scale mutagenesis screen for mutations that disrupt the M lineage and the sex myoblast (SM) sublineage. We isolated and characterized mutations that result in a failure of proper SM fate specification and SM migration and showed that the corresponding genes encode Ppa-sem-4 and Ppa-egl-17, respectively. Ppa-sem-4 mutants have additional defects in the specification of the vulva precursor cells P(5, 7).p and experimental studies in the Ppa-egl-17 mutant background indicate a complex set of gonad-dependent and gonad-independent mechanisms required for SM migration. Mutations in Cel-sem-4 and Cel-egl-17 cause similar defects. Thus, the molecular mechanisms of SM cell specification and migration are conserved between P. pacificus and C. elegans.     

The embryonic cell lineage of the nematode Rhabditophanes sp

One of the unique features of the model organism Caenorhabditis elegans is its invariant development, where a stereotyped cell lineage generates a fixed number of cells with a fixed cell type. It remains unclear how embryonic development evolved within the nematodes to give rise to the complex, invariant cell lineage of C. elegans. Therefore, we determined the embryonic cell lineage of the nematode, Rhabditophanes sp. (family Alloionematidae) and made detailed cell-by-cell comparison with the known cell lineages of C. elegans, Pellioditis marina and Halicephalobus gingivalis. This gave us a unique data set of four embryonic cell lineages, which allowed a detailed comparison between these cell lineages at the level of each individual cell. This lineage comparison revealed a similar complex polyclonal fate distribution in all four nematode species (85% of the cells have the same fate). It is striking that there is a conservation of a 'C. elegans' like polyclonal cell lineage with strong left-right asymmetry. We propose that an early symmetry-breaking event in nematodes of clade IV-V is a major developmental constraint which shapes their asymmetric cell lineage.     

Regulation of anchor cell invasion and uterine cell fates by the egl-43 Evi-1 proto-oncogene in Caenorhabditis elegans

Cell invasion is a tightly controlled process occurring during development and tumor progression. The nematode Caenorhabditis elegans serves as a genetic model to study cell invasion during normal development. In the third larval stage, the anchor cell in the somatic gonad first induces and then invades the adjacent epidermal vulval precursor cells. The homolog of the Evi-1 oncogene, egl-43, is necessary for basement membrane destruction and anchor cell invasion. egl-43 is part of a regulatory network mediating cell invasion downstream of the fos-1 proto-oncogene. In addition, EGL-43 is required to specify the cell fates of ventral uterus cells downstream of or in parallel with LIN-12 NOTCH. Comparison with mammalian Evi-1 suggests a conserved pathway controlling cell invasion and cell fate specification.     

Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.     

cki-1 links cell division and cell fate acquisition in the C. elegans somatic gonad

The formation of a complex multicellular organism requires the precise specification of many diverse cell types at the correct time and position throughout development. This may be achieved by coordinating cell fate specification processes with progression through the cell cycle. Here, we show that the extra distal tip cells (DTCs) associated with the loss of cki-1, a Caenorhabditis elegans homologue of the cyclin-dependent kinase inhibitor p27, do not arise from duplications of pre-existing DTCs, but that they are formed from another cell type within the somatic gonad. Results from our laser microsurgery experiments suggest that the extra DTCs are caused by aberrant somatic gonadal precursor cell divisions in the absence of cki-1, resulting in abnormal daughter cell fates. cki-1(RNAi) animals also possess extra anchor cells and ectopic gonad arms with variable sheath cell numbers and positioning. In addition, cki-1(RNAi) animals display an endomitotic oocyte (Emo) phenotype. Our results uncover a novel role of this CKI in cell fate acquisition, either by directly influencing specification, or through a more conventional role in appropriately linking cell cycle phase with this process.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans.

Vulval development in the nematode Caenorhabditis elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, we investigated the role of Rac signaling during each phase of vulval development. We show that the Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. We discuss the similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

Embryo-patterning genes and reinforcement cues determine cell fate in theArabidopsis thalianaroot

The majority of plant organs arise from groups of continuously dividing cells, the meristems. Little is known about mechanisms of cell specification in meristems. Within theArabidopsisroot meristem, the fate of every cell can be predicted accurately, and the origin of these cells during the formation of the embryonic root primordium is known. Laser ablations reveal that, despite the regularity in cell lineage, position remains important to reinforce cell specification. Genetic analysis has revealed that many genes involved in the specification of the main cell types in the root act early, during embryogenesis, and an important question is whether the same or other genes are involved in the reinforcement of specification. Sub-specification of cell types, as exemplified by epidermal root hair cell specification, involves two pathways, one of which may act to reinforce earlier patterning events mediated by the other.     

mig-5/Dsh controls cell fate determination and cell migration in C. elegans

Cell fate determination and cell migration are two essential events in the development of an organism. We identify mig-5, a Dishevelled family member, as a gene that regulates several cell fate decisions and cell migrations that are important during C. elegans embryonic and larval development. In offspring from mig-5 mutants, cell migrations are defective during hypodermal morphogenesis, QL neuroblast migration, and the gonad arm migration led by the distal tip cells (DTCs). In addition to abnormal migration, DTC fate is affected, resulting in either an absent or an extra DTC. The cell fates of the anchor cell in hermaphrodites and the linker cells in the male gonad are also defective, often resulting in the cells adopting the fates of their sister lineage. Moreover, 2 degrees vulval precursor cells occasionally adopt the 3 degrees vulval cell fate, resulting in a deformed vulva, and the P12 hypodermal precursor often differentiates into a second P11 cell. These defects demonstrate that MIG-5 is essential in determining proper cell fate and cell migration throughout C. elegans development.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, as neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. Here we show that the C. elegansdsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. We suggest that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ.     

Identification and lineage tracing of two populations of somatic gonadal precursors in medaka embryos

The gonad contains two major cell lineages, germline and somatic cells. Little is known, however, about the somatic gonadal cell lineage in vertebrates. Using fate mapping studies and ablation experiments in medaka fish (Oryzias latipes), we determined that somatic gonadal precursors arise from the most posterior part of the sdf-1a expression domain in the lateral plate mesoderm at the early segmentation stage; this region has the properties of a gonadal field. Somatic gonadal precursors in this field, which continuously express sdf-1a, move anteriorly and medially to the prospective gonadal area by convergent movement. By the stage at which these somatic gonadal precursors have become located adjacent to the embryonic body, the precursors no longer replace the surrounding lateral plate mesoderm, becoming spatially organized into two distinct populations. We further show that, prior to reaching the prospective gonadal area, these populations can be distinguished by expression of either ftz-f1 or sox9b. These results clearly indicate that different populations of gonadal precursors are present before the formation of a single gonadal primordium, shedding new light on the developmental processes of somatic gonadal cell and subsequent sex differentiation.     

tcl-2 encodes a novel protein that acts synergistically with Wnt signaling pathways in C. elegans

Mutations in tcl-2 cause defects in the specification of the fates of the descendants of the TL and TR blast cells, whose polarity is regulated by lin-44/Wnt and lin-17/frizzled, during Caenorhabditis elegans development. In wild-type animals, POP-1/TCF/LEF, is asymmetrically distributed to the T cell daughters, resulting in a higher level of POP-1 in the nucleus of the anterior daughter. The POP-1 asymmetric distribution is controlled by lin-44 and lin-17. However, in tcl-2 mutants, POP-1 is equally distributed to T cell daughters as is observed in lin-17 mutants, indicating that, like lin-17, tcl-2 functions upstream of pop-1. In addition, tcl-2 mutations cause defects in the development of the gonad and the specification of fate of the posterior daughter of the P12 cell, both of which are controlled by the Wnt pathway. Double mutant analyses indicate that tcl-2 can act synergistically with the Wnt pathway to control gonad development as well as P12 descendant cell fate specification. tcl-2 encodes a novel protein. A functional tcl-2::gfp construct was weakly expressed in the nuclei of the T cell and its descendants. Our results suggest that tcl-2 functions with Wnt pathways to control T cell fate specification, gonad development, and P12 cell fate specification.