The effect of developmental arrest on stem/progenitor cells and developmental pathways
The complex process of animal development occurs in the context of the surrounding environment. Species from nematodes to vertebrates can undergo a diapause stage where developmental progression is arrested in order to survive adverse environmental conditions. We use the powerful Caenorhabditis elegans model species to study two aspects of diapause and its effect on development.
- During diapause, how do stem and progenitor cells “remember” their precise place in their developmental program, neither differentiating prematurely, nor losing their tissue identity? Many of the same genetic pathways that regulate mammalian stem cell quiescence (reversable cell-cycle arrest) and stem cell maintenance also regulate diapause and stem/progenitor cells in C. elegans, making our work applicable to human health. This work has been supported by the NIH.
- How are developmental pathways altered by diapause to allow similar developmental outcomes in diapause and non-diapause animals? In C. elegans, some genes required for wild-type development in non-diapause contexts are dispensable after diapause, indicating that there are separate pathways that operate after dauer and/or that core developmental pathways are modulated. Projects exploring how core developmental pathways are modulated and identifying new genes that act after diapause are currently funded by the NSF.
Why use C. elegans as a model?
Caenorhabditis elegans is a free-living, microscopic nematode. C. elegans is a popular model system due to its simplicity, ease of handling, and amenability to genetic analysis. Because genetic pathways are so well conserved from worms to mammals, much of what is discovered in the worm is relevant to human disease. To date, three Nobel Prizes have been awarded for C. elegans research (2002, 2006, 2008)
One major advantage to using C. elegans as a model system is their essentially invariant cell lineage. Adult C. elegans hermaphrodites have precisely 959 somatic cells that arise from nearly identical cell divisions in each individual. Because C. elegans is transparent, each cell division has been observed and documented. Furthermore, fluorescent cell fate markers highlight particular cells at particular stages in their developmental program. Thus, cell fate can be followed at the single cell level, and any deviations from wild-type can be readily observed.
C. elegans as a stem cell model
Adult stem cells typically remain quiescent for long periods, dividing only as needed to maintain tissue integrity. In order to contribute to tissue renewal, stem cells remain multipotent and plastic, meaning they can give rise to multiple, specific cell-types. Certain C. elegans progenitor cells share similar properties, including quiescence and multipotency. C. elegans larvae develop continuously through four larval stages punctuated by molts. During each stage, progenitor cells divide and contribute to the growth of the worm. In adverse environments (crowding, insufficient food, higher temperatures), C. elegans larvae can arrest their development in the stress-resistant dauer larva stage. Dauer formation occurs immediately after the second larval molt. Since larval development is incomplete at this point, dauer larvae possess progenitor cells with the capacity to give rise to various tissues, including the lateral hypodermis (skin), and the vulva, among others. All cells within dauer larvae are quiescent throughout dauer arrest, which can last from hours to months. This time span is remarkable given that the normal C. elegans lifespan is only a few weeks. If dauer larvae encounter favorable environments they will recover, complete development normally, and live a normal lifespan on top of the time they spent in dauer.
Lateral hypodermis: post-dauer timing and microRNA activity
One cell-type used as a stem-cell model is the lateral hypodermal seam cells that form part of the worm skin. These cells divide each larval stage in a characteristic pattern that includes self-renewal as well as the production of other skin cell types. The stage at which each cell division pattern occurs is called “stage-specific cell fate”. Stage-specific cell fate is controlled by a gene network called “heterochronic”. Mutations in heterochronic genes cause two types of defect: precocious and reiterative. Often, the precocious genes encode transcription factors that specify early cell fate, whereas reiterative genes encode microRNAs that downregulate expression of the transcription factors.
Surprisingly, many heterochronic genes that are important during continuous development are dispensable after dauer. Some of the differences between phenotypes during continuous and post-dauer development can be explained by modulation of microRNA activity, making dauer an excellent system to probe the biological relevance of microRNA pathways and their regulation. We are interested in unraveling the gene network that operates in post-dauer worms, including microRNA pathways. To accomplish this we are probing the role of candidate genes as well as performing unbiased genetic screens for mutants defective in post-dauer timing.
Vulval precursor cells: signal transduction pathways and cell fate plasticity
A second cell type studied in the Karp lab is vulval precursor cells. These six cells are born in the L1 stage and remain multipotent until specification occurs during the L3 stage. During dauer, a mechanism exists to maintain plasticity by blocking the activity of signal transduction pathways, and erasing prior specification. This mechanism involves DAF-16, the C. elegans ortholog of FOXO transcription factors, proteins with conserved roles in longevity and stress-resistance. We are interested in elucidating the mechanisms by which daf-16 promotes cell fate plasticity during dauer, including discovering the genes that DAF-16 regulates controlling plasticity.