Beth Thompson

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF (‘fem-3 mRNA binding factor’), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family (‘Pumilio and FBF’) of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.

FBF and Its Dual Control of gld-1 Expression in the Caenorhabditis elegans Germline

FBF, a PUF RNA-binding protein, is a key regulator of the mitosis/meiosis decision in the Caenorhabditis elegans germline. Genetically, FBF has a dual role in this decision: it maintains germ cells in mitosis, but it also facilitates entry into meiosis. In this article, we explore the molecular basis of that dual role. Previous work showed that FBF downregulates gld-1 expression to promote mitosis and that the GLD-2 poly(A) polymerase upregulates gld-1 expression to reinforce the decision to enter meiosis. Here we ask whether FBF can act as both a negative regulator and a positive regulator of gld-1 expression and also investigate its molecular mechanisms of control. We first show that FBF co-immunoprecipitates with gld-1 mRNA, a result that complements previous evidence that FBF directly controls gld-1 mRNA. Then we show that FBF represses gld-1 expression, that FBF physically interacts with the CCF-1/Pop2p deadenylase and can stimulate deadenylation in vitro, and that CCF-1 is partially responsible for maintaining low GLD-1 in the mitotic region. Finally, we show that FBF can elevate gld-1 expression, that FBF physically interacts with the GLD-2 poly(A) polymerase, and that FBF can enhance GLD-2 poly(A) polymerase activity in vitro. We propose that FBF can affect polyadenylation either negatively by its CCF-1 interaction or positively by its GLD-2 interaction.

Dose-dependent control of proliferation and sperm specification by FOG-1/CPEB.

RNA-binding proteins control germline development in metazoans. This work focuses on control of the C. elegans germline by two RNA-binding proteins: FOG-1, a CPEB homolog; and FBF, a PUF family member. Previous studies have shown that FOG-1 specifies the sperm fate and that FBF promotes proliferation. Here, we report that FOG-1 also promotes proliferation. Whereas fbf-1 fbf-2 double mutants make ∼120 germ cells, fog-1; fbf-1 fbf-2 triple mutants make only ∼10 germ cells. The triple mutant germline divides normally until early L2, when germ cells prematurely enter meiosis and begin oogenesis. Importantly, fog-1/+; fbf-1 fbf-2 animals make more germ cells than fbf-1 fbf-2 double mutants, demonstrating that one dose of wild-type fog-1 promotes proliferation more effectively than two doses – at least in the absence of FBF. FOG-1 protein is barely detectable in proliferating germ cells, but abundant in germ cells destined for spermatogenesis. Based on fog-1 dose effects, together with the gradient of FOG-1 protein abundance, we suggest that low FOG-1 promotes proliferation and high FOG-1 specifies spermatogenesis. FBF binds specifically to regulatory elements in the fog-1 3′UTR, and FOG-1 increases in animals lacking FBF. Therefore, FBF represses fog-1 expression. We suggest that FBF promotes continued proliferation, at least in part, by maintaining FOG-1 at a low level appropriate for proliferation. The dose-dependent control of proliferation and cell fate by FOG-1 has striking parallels with Xenopus CPEB, suggesting a conserved mechanism in animal development.

Translational biology: from Arabidopsis flowers to grass inflorescence architecture.

One of the key events in plant development is the initiation of lateral organs from the flanks of the meristem. In grasses, the inflorescence meristem (IM) reiteratively initiates a series of lateral meristems with slightly different fates. Our understanding of the genes and networks that regulate

Meristem identity and phyllotaxis in inflorescence development

Inflorescence morphology is incredibly diverse. This diversity of form has been a fruitful source of inquiry for plant morphologists for more than a century. Work in the grasses (Poaceae), the tomato family (Solanaceae), and Arabidopsis thaliana (Brassicaceae) has led to a richer understanding of the molecular genetics underlying this diversity. The character of individual meristems, a combination of the number (determinacy) and nature (identity) of the products a meristem produces, is key in the development of plant form. A framework that describes inflorescence development in terms of shifting meristem identities has emerged and garnered empirical support in a number of model systems. We discuss this framework and highlight one important aspect of meristem identity that is often considered in isolation, phyllotaxis. Phyllotaxis refers to the arrangement of lateral organs around a central axis. The development and evolution of phyllotaxis in the inflorescence remains underexplored, but recent work analyzing early inflorescence development in the grasses identified an evolutionary shift in primary branch phyllotaxis in the Pooideae. We discuss the evidence for an intimate connection between meristem identity and phyllotaxis in both the inflorescence and vegetative shoot, and touch on what is known about the establishment of phyllotactic patterns in the meristem. Localized auxin maxima are instrumental in determining the position of lateral primordia. Upstream factors that regulate the position of these maxima remain unclear, and how phyllotactic patterns change over the course of a plants lifetime and evolutionary time, is largely unknown. A more complete understanding of the molecular underpinnings of phyllotaxis and architectural diversity in inflorescences will require capitalizing on the extensive resources available in existing genetic systems, and developing new model systems that more fully represent the diversity of plant morphology.

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein–Protein Interactions

Protein–protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PIL) and AP3-like (AP3L) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PIL protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PIL homodimerization is an anomaly or indicative of broader trends, we characterized PIL dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PIL homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PIL homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PIL protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PIL dimerization activity. Furthermore, ectopic expression of a Joinvillea PIL homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner.

The dicer-like1 Homolog fuzzy tassel Is Required for the Regulation of Meristem Determinacy in the Inflorescence and Vegetative Growth in Maize

The maize fuzzy tassel mutant is the first dcl1 mutant reported in a plant other than Arabidopsis and enables functional analysis of specific miRNAs and their targets. Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles.

Analysis of the Maize dicer-like1 Mutant, fuzzy tassel, Implicates MicroRNAs in Anther Maturation and Dehiscence

Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.

Genetic and Hormonal Regulation of Maize Inflorescence Development

Abstract Plant development is determined by the activity of meristems, pools of undifferentiated cells that generate organs throughout the life of the plant. Inflorescence morphology is determined by the position and activity of meristems that form during reproductive development. Grass inflorescences are complex, highly branched structures that initiate higher order meristems before the floral meristems, which ultimately produce the floral organs. Maize produces two inflorescences: the tassel is formed at the apex of the plant and bears male flowers and the ear is formed in the axil of a leaf and bears female flowers. Despite their distinct morphologies at maturity, the tassel and ear arise from strikingly similar inflorescence primordia and their patterning depends largely on the same set of developmental regulators. Many of these developmental regulators have been identified through mutant analysis and cloning efforts, and include multiple transcription factors, microRNAs and plant hormones. This chapter focuses on the molecular mechanisms that regulate the maintenance, identity, and activity of meristems in maize inflorescences, as well as floral development and sex determination.

19 Translational Control in Development

Development requires the coordinated expression of selected genes at specific times and in specific cells. Such regulated expression controls the establishment of embryonic axes, the existence of stem cells, and the specification of individual cell fates. Translational control has a key role in this regulation. It is particularly important during early embryogenesis and in the germ line, where transcription is typically quiescent; there, control of translation and mRNA stability are the primary ways to regulate patterns of protein synthesis. Yet, translational regulation continues throughout development and in somatic tissues. In this chapter, we view translational control from a developmental perspective. We discuss four major interfaces at which developmental biology meets molecular regulatory mechanisms: molecular switches, gradients, combinatorial control, and networks. These areas were chosen because they bear on fundamental processes of development. We emphasize instances in which sequence-specific regulatory factors control particular mRNAs, and we do not cover the role of general translation factors (e.g., eIF4E and eIF2α) on growth and differentiation (Chapter 4). MECHANISMS OF TRANSLATIONAL CONTROL: A PRIMER Translation is a multistep process and can be divided into three phases: initiation, elongation, and termination. In principle, translational control can be exerted in any of these phases. We focus in this chapter on initiation, which appears to be the most common point of control during development. Translational initiation involves more than 20 proteins, multiple biochemical complexes, and a series of separable steps (Chapter 4). A complex containing eukaryotic initiation factor 4E (eIF4E) (cap-binding protein) and eIF4G is crucial. At...