A temporal sequence of heterochronic gene activities promotes stage-specific developmental events in C. elegans.

The heterochronic genes of the nematode Caenorhabditis elegans control the succession of postembryonic developmental events. The four core heterochronic genes lin-14, lin-28, hbl-1, and lin-41 act in a sequence to specify cell fates specific to each of the four larval stages. It was previously shown that lin-14 has two activities separated in time that promote L1 and L2 developmental events, respectively. Using the auxin-inducible degron system, we find that lin-28 and hbl-1 each have two activities that control L2 and L3 events which are also separated in time. Relative to events they control, both lin-28 and hbl-1 appear to act just prior to or concurrently with events of the L2. Relative to each other, lin-28 and hbl-1 appear to act simultaneously. By contrast, the lin-14 activity controlling L2 events precedes those of lin-28 and hbl-1 controlling the same events, suggesting lin-14's regulation of lin-28 is responsible for the delay. Likewise, the activities of lin-28 and hbl-1 controlling L3 fates act well in advance of those fates, suggesting a similar regulatory gap. lin-41 acts early in the L3 to affect fates of the L4, although it was not possible to determine whether it too has two temporally separated activities. We also uncovered a feedback phenomenon that prevents the reactivation of heterochronic gene activity late in development after it has been down-regulated. This study places the heterochronic gene activities into a timeline of postembryonic development relative to one another and to the developmental events whose timing they control.

Orthologs of the Caenorhabditis elegans heterochronic genes have divergent functions in Caenorhabditis briggsae.

The heterochronic genes of Caenorhabditis elegans comprise the best-studied pathway controlling the timing of tissue and organ formation in an animal. To begin to understand the evolution of this pathway and the significance of the relationships among its components, we characterized 11 Caenorhabditis briggsae orthologs of C. elegans heterochronic genes. Using CRISPR/Cas9, we made a variety of alleles and found that several mutant phenotypes differ in significant ways from those of C. elegans. Although most mutant orthologs displayed defects in developmental timing, their phenotypes could differ in which stages were affected, the penetrance and expressivity of the phenotypes, or by having additional pleiotropies that were not obviously connected to developmental timing. However, when examining pairwise epistasis and synergistic relationships, we found those paralleled the known relationships between their C. elegans orthologs, suggesting that the arrangements of these genes in functional modules are conserved, but the modules' relationships to each other and/or to their targets has drifted since the time of the species' last common ancestor. Furthermore, our investigation has revealed a relationship between this pathway to other aspects of the animal's growth and development, including gonad development, which is relevant to both species.

An engineered, orthogonal auxin analog/AtTIR1(F79G) pairing improves both specificity and efficacy of the auxin degradation system in Caenorhabditis elegans.

The auxin-inducible degradation system in C. elegans allows for spatial and temporal control of protein degradation via heterologous expression of a single Arabidopsis thaliana F-box protein, transport inhibitor response 1 (AtTIR1). In this system, exogenous auxin (Indole-3-acetic acid; IAA) enhances the ability of AtTIR1 to function as a substrate recognition component that adapts engineered degron-tagged proteins to the endogenous C. elegans E3 ubiquitin ligases complex [SKR-1/2-CUL-1-F-box (SCF)], targeting them for degradation by the proteosome. While this system has been employed to dissect the developmental functions of many C. elegans proteins, we have found that several auxin-inducible degron (AID)-tagged proteins are constitutively degraded by AtTIR1 in the absence of auxin, leading to undesired loss-of-function phenotypes. In this manuscript, we adapt an orthogonal auxin derivative/mutant AtTIR1 pair [C. elegans AID version 2 (C.e.AIDv2)] that transforms the specificity of allosteric regulation of TIR1 from IAA to one that is dependent on an auxin derivative harboring a bulky aryl group (5-Ph-IAA). We find that a mutant AtTIR1(F79G) allele that alters the ligand-binding interface of TIR1 dramatically reduces ligand-independent degradation of multiple AID*-tagged proteins. In addition to solving the ectopic degradation problem for some AID-targets, the addition of 5-Ph-IAA to culture media of animals expressing AtTIR1(F79G) leads to more penetrant loss-of-function phenotypes for AID*-tagged proteins than those elicited by the AtTIR1-IAA pairing at similar auxin analog concentrations. The improved specificity and efficacy afforded by the mutant AtTIR1(F79G) allele expand the utility of the AID system and broaden the number of proteins that can be effectively targeted with it.

The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

The RNA-binding protein LIN28 is known to regulate cell fate, tissue growth, and pluripotency; however, a unified understanding of its role at the cellular level has not been achieved. Here, we address its developmental activity in mammalian postnatal neurogenesis. Constitutive expression of LIN28 in progenitor cells of the mouse subventricular zone (SVZ) caused several distinct effects: 1) the number of differentiated neurons in the olfactory bulb was dramatically reduced, whereas the relative abundance of 2 neuronal subtypes was significantly altered, 2) the population of proliferating neural progenitors in the SVZ was reduced, whereas the proportion of neuroblasts was increased, and 3) the number of astrocytes was reduced, occasionally causing them to appear early. Thus, LIN28 acts at a poststem cell/predifferentiation step, and its continuous expression caused a precocious phenotype unlike in other experimental systems. Furthermore, for the first time in a vertebrate system, we separate the majority of the biologic role of LIN28 from its known activity of blocking the microRNA let-7 by using a circular RNA sponge. We find that although LIN28 has a multifaceted role in the number and types of cells produced during postnatal neurogenesis, it appears that its action through let-7 is responsible for only a fraction of these effects.-Romer-Seibert, J. S., Hartman, N. W., Moss, E. G. The RNA-binding protein LIN28 controls progenitor and neuronal cell fate during postnatal neurogenesis.

Stage-Specific Timing of the microRNA Regulation of lin-28 by the Heterochronic Gene lin-14 in Caenorhabditis elegans.

In normal development, the order and synchrony of diverse developmental events must be explicitly controlled. In the nematode Caenorhabditis elegans, the timing of larval events is regulated by hierarchy of proteins and microRNAs (miRNAs) known as the heterochronic pathway. These regulators are organized in feedforward and feedback interactions to form a robust mechanism for specifying the timing and execution of cell fates at successive stages. One member of this pathway is the RNA binding protein LIN-28, which promotes pluripotency and cell fate decisions in successive stages. Two genetic circuits control LIN-28 abundance: it is negatively regulated by the miRNA lin-4, and positively regulated by the transcription factor LIN-14 through a mechanism that was previously unknown. In this report, we used animals that lack lin-4 to elucidate LIN-14's activity in this circuit. We demonstrate that three let-7 family miRNAs-miR-48, miR-84, and miR-241-inhibit lin-28 expression. Furthermore, we show genetically that these miRNAs act between lin-14 and lin-28, and that they comprise the pathway by which lin-14 positively regulates lin-28 We also show that the lin-4 family member mir-237, also regulates early cell fates. Finally, we show that the expression of these miRNAs is directly inhibited by lin-14 activity, making them the first known targets of lin-14 that act in the heterochronic pathway.

Lin28 promotes the proliferative capacity of neural progenitor cells in brain development.

Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development.

Cell-intrinsic timing in animal development.

UNLABELLED: In certain instances we can witness cells controlling the sequence of their behaviors as they divide and differentiate. Striking examples occur in the nervous systems of animals where the order of differentiated cell types can be traced to internal changes in their progenitors. Elucidating the molecular mechanisms underlying such cell fate succession has been of interest for its role in generating cell type diversity and proper tissue structure. Another well-studied instance of developmental timing occurs in the larva of the nematode Caenorhabditis elegans, where the heterochronic gene pathway controls the succession of a variety of developmental events. In each case, the identification of molecules involved and the elucidation of their regulatory relationships is ongoing, but some important factors and dynamics have been revealed. In particular, certain homologs of worm heterochronic factors have been shown to work in neural development, alerting us to possible connections among these systems and the possibility of universal components of timing mechanisms. These connections also cause us to consider whether cell-intrinsic timing is more widespread, regardless of whether multiple differentiated cell types are produced in any particular order. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.

Developmental transitions in C. elegans larval stages.

Molecular mechanisms control the timing, sequence, and synchrony of developmental events in multicellular organisms. In Caenorhabditis elegans, these mechanisms are revealed through the analysis of mutants with "heterochronic" defects: cell division or differentiation patterns that occur in the correct lineage, but simply at the wrong time. Subsets of cells in these mutants thus express temporal identities normally restricted to a different life stage. A seminal finding arising from studies of the heterochronic genes was the discovery of miRNAs; these tiny miRNAs are now a defining feature of the pathway. A series of sequentially expressed miRNAs guide larval transitions through stage-specific repression of key effector molecules. The wild-type lineage patterns are executed as discrete modules programmed between temporal borders imposed by the molting cycles. How these successive events are synchronized with the oscillatory molting cycle is just beginning to come to light. Progression through larval stages can be specifically, yet reversibly, halted in response to environmental cues, including nutrient availability. Here too, heterochronic genes and miRNAs play key roles. Remarkably, developmental arrest can, in some cases, either mask or reveal timing defects associated with mutations. In this chapter, we provide an overview of how the C. elegans heterochronic gene pathway guides developmental transitions during continuous and interrupted larval development.

Fetal deficiency of lin28 programs life-long aberrations in growth and glucose metabolism.

LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here, we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO could be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.

Lin28a regulates germ cell pool size and fertility.

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males, the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild-type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.

lin-28 controls the succession of cell fate choices via two distinct activities.

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

LIN28 alters cell fate succession and acts independently of the let-7 microRNA during neurogliogenesis in vitro.

LIN28 is an RNA-binding protein that is expressed in many developing tissues. It can block let-7 (Mirlet7) microRNA processing and help promote pluripotency. We have observed LIN28 expression in the developing mouse neural tube, colocalizing with SOX2, suggesting a role in neural development. To better understand its normal developmental function, we investigated LIN28 activity during neurogliogenesis in vitro, where the succession of neuronal to glial cell fates occurs as it does in vivo. LIN28 expression was high in undifferentiated cells, and was downregulated rapidly upon differentiation. Constitutive LIN28 expression caused a complete block of gliogenesis and an increase in neurogenesis. LIN28 expression was compatible with neuronal differentiation and did not increase proliferation. LIN28 caused significant changes in gene expression prior to any effect on let-7, notably on Igf2. Furthermore, a mutant LIN28 that permitted let-7 accumulation was still able to completely block gliogenesis. Thus, at least two biological activities of LIN28 are genetically separable and might involve distinct mechanisms. LIN28 can differentially promote and inhibit specific fates and does not function exclusively by blocking let-7 family microRNAs. Importantly, the role of LIN28 in cell fate succession in vertebrate cells is analogous to its activity as a developmental timing regulator in C. elegans.

Localization of the developmental timing regulator Lin28 to mRNP complexes, P-bodies and stress granules.

Lin28 is a conserved cytoplasmic protein with an unusual pairing of RNA-binding motifs: a cold shock domain and a pair of retroviral-type CCHC zinc fingers. In the nematode C. elegans, it is a regulator of developmental timing. In mammals, it is abundant in diverse types of undifferentiated cells. However, its molecular function is unknown. In pluripotent mammalian cells, Lin28 is observed in RNase-sensitive complexes with poly(A)-binding protein, and in polysomal fractions of sucrose gradients, suggesting it is associated with translating mRNAs. Upon cellular stress, Lin28 locates to stress granules, which contain non-translating mRNA complexes. However, Lin28 also localizes to cytoplasmic processing bodies, or P-bodies, sites of mRNA degradation and microRNA regulation, consistent with it acting to regulate mRNA translation or stability. Mutational analysis shows that Lin28's conserved RNA binding domains cooperate to put Lin28 in mRNPs, but that only the CCHC domain is required for localization to P-bodies. When both RNA-binding domains are mutated, Lin28 accumulates in the nucleus, suggesting that it normally shuttles from nucleus to cytoplasm bound to RNA. These studies are consistent with a model in which Lin28 binds mRNAs in the nucleus and accompanies them to ribosomes and P-bodies. We propose that Lin28 influences the translation or stability of specific mRNAs during differentiation.

Heterochronic genes and the nature of developmental time.

Timing is a fundamental issue in development, with a range of implications from birth defects to evolution. In the roundworm Caenorhabditis elegans, the heterochronic genes encode components of a molecular developmental timing mechanism. This mechanism functions in diverse cell types throughout the animal to specify cell fates at each larval stage. MicroRNAs play an important role in this mechanism by stage-specifically repressing cell-fate regulators. Recent studies reveal the surprising complexity surrounding this regulation--for example, a positive feedback loop may make the regulation more robust, and certain components of the mechanism are expressed in brief periods at each stage. Other factors reveal the potential for important roles of steroid hormones and targeted proteolysis. Investigation of the heterochronic genes has revealed a mechanism composed of precisely timed switches linked to discrete developmental stages. Timing is a dimension of developmental regulation that may be difficult to witness in vertebrates because developmental stages are not as discrete as in C. elegans, each tissue is likely to be independently regulated. Homologs of certain heterochronic genes of vertebrates show temporally regulated expression patterns, and may ultimately reveal timing mechanisms not previously known to exist.

Lin-28 binds IGF-2 mRNA and participates in skeletal myogenesis by increasing translation efficiency.

Lin-28 is a highly conserved, RNA-binding, microRNA-regulated protein that is involved in regulation of developmental timing in Caenorhabditis elegans. In mammals, Lin-28 is stage-specifically expressed in embryonic muscle, neurons, and epithelia, as well as in embryonic carcinoma cells, but is suppressed in most adult tissues, with the notable exception of skeletal and cardiac muscle. The specific function and mechanism of action of Lin-28 are not well understood. Here we used loss-of-function and gain-of-function assays in cultured myoblasts to show that expression of Lin-28 is essential for skeletal muscle differentiation in mice. In order to elucidate the specific function of Lin-28, we used a combination of biochemical and functional assays, which revealed that, in differentiating myoblasts, Lin-28 binds to the polysomes and increases the efficiency of protein synthesis. An important target of Lin-28 is IGF-2, a crucial growth and differentiation factor for muscle tissue. Interaction of Lin-28 with translation initiation complexes in skeletal myoblasts and in the embryonic carcinoma cell line P19 was confirmed by localization of Lin-28 to the stress granules, temporary structures that contain stalled mRNA-protein translation complexes. Our results unravel novel mechanisms of translational regulation in skeletal muscle and suggest that Lin-28 performs the role of "translational enhancer" in embryonic and adult cells and tissues.

The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin.

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. We also show that lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.

Temporally regulated expression of Lin-28 in diverse tissues of the developing mouse.

The gene lin-28 was originally identified through a mutant of the nematode Caenorhabditis elegans displaying defects in developmental timing. It is expressed stage-specifically in tissues throughout the animal and is required for cell fates to be expressed at the appropriate stage of larval development. lin-28 encodes a cytoplasmic protein with a unique pairing of RNA-binding motifs. Diverse animals possess Lin-28 homologues and mouse Lin-28 is expressed in embryos, embryonic stem cells and embryonal carcinoma cells, but not in some differentiated cell types. To assess whether mammalian Lin-28 may function as a developmental timing regulator, we examined adult and embryonic tissues of the mouse for its expression. We observed Lin-28 protein in many diverse tissues of the embryo through the period of organogenesis and that it persists in some tissues in the adult. In addition to an overall down-regulation during embryogenesis, in at least two tissues Lin-28 expression shows temporal regulation, as opposed to cell type or tissue-specific regulation: in the developing bronchial epithelium, where it is present in the developing lung and absent in the adult, and in a subset of cells developing along the crypt-villus axis of the intestine. Interestingly, unlike epithelia, cardiac and skeletal muscle continuously express Lin-28, suggesting an ongoing need for its activity there. We also observed that Lin-28 expression is repressed during the retinoic acid-induced differentiation of mouse P19 cells into neuronal cells, suggesting that down-regulation of Lin-28 in some tissues may occur in response to hormonal signals that govern development.

RNA repair: damage control.

RNA in a cell is subject to many of the same insults as DNA. RNA damage can induce apoptosis and may be exploited for anti-cancer chemotherapy. It is a surprise, however, to learn that cells may repair RNA damage, suggesting a far greater significance of RNA in genotoxic stress.