Heterochronic Gene Pathway Research Articles

Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing.

The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

Cell-intrinsic timing in animal development.

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. The authors have declared no conflicts of interest for this article.

RACK-1 regulates let-7 microRNA expression and terminal cell differentiation in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development.

The Heterochronic Genes lin-28a and lin-28b Play an Essential and Evolutionarily Conserved Role in Early Zebrafish Development

The Caenorhabditis elegans heterochronic gene pathway, which consists of a set of regulatory genes, plays an important regulatory role in the timing of stage-specific cell lineage development in nematodes. Research into the heterochronic gene pathway gave rise to landmark microRNA (miRNA) studies and showed that these genes are important in stem cell and cancer biology; however, their functions in vertebrate development are largely unknown. To elucidate the function of the heterochronic gene pathway during vertebrate development, we cloned the zebrafish homologs of the C. elegans let-7 miRNA-binding protein, Lin-28, and analyzed their function in zebrafish development. The zebrafish genome contains two Lin28-related genes, lin-28a and lin-28b. Similar to mammalian Lin28 proteins, both zebrafish Lin-28a and Lin-28b have a conserved cold-shock domain and a pair of CCHC zinc finger domains, and are ubiquitously expressed during early embryonic development. In a reciprocal fashion, the expression of downstream heterochronic genes, let-7 and lin-4/miR-125 miRNA, occurred subsequent to lin-28 expression. The knockdown of Lin-28a or Lin-28b function by morpholino microinjection into embryos resulted in severe cell proliferation defects during early morphogenesis. We found that the expression of let-7 miRNA was upregulated and its downstream target gene, lin-41, was downregulated in these embryos. Interestingly, the expression of miR-430, a key regulator of maternal mRNA decay, was downregulated in lin-28a and lin-28b morphant embryos, suggesting a role for Lin-28 in the maternal-to-zygotic transition in zebrafish. Taken together, our results suggest an evolutionarily conserved and pivotal role of the heterochronic gene pathway in early vertebrate embryogenesis.

Heterochronic genes in plant evolution and development

Evolution of morphology includes evolutionary shifts of developmental processes in space or in time. Heterochronic evolution is defined as a temporal shift. The concept of heterochrony has been very rewarding to investigators of both animal and plant developmental evolution, because it has strong explanatory power when trying to understand morphological diversity. While for animals, extensive literature on heterochrony developed along with the field of evolution of development, in plants the concept has been applied less often and is less elaborately developed. Yet novel genetic findings highlight heterochrony as a developmental and evolutionary process in plants. Similar to what has been found for the worm Caenorhabditis, a heterochronic gene pathway controlling developmental timing has been elucidated in flowering plants. Two antagonistic microRNA’s miR156 and miR172 target two gene families of transcription factors, SQUAMOSA PROMOTOR BINDING PROTEIN-LIKE and APETALA2-like, respectively. Here, we propose that this finding now allows the molecular investigation of cases of heterochronic evolution in plants. We illustrate this point by examining microRNA expression patterns in the Antirrhinum majus incomposita and choripetala heterochronic mutants. Some of the more beautiful putative cases of heterochronic evolution can be found outside flowering plants, but little is known about the extent of conservation of this flowering plant pathway in other land plants. We show that the expression of an APETALA2-like gene decreases with age in a fern species. This contributes to the idea that ferns share some heterochronic gene functions with flowering plants.

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.

The nuclear receptor gene nhr-25 plays multiple roles in the Caenorhabditis elegans heterochronic gene network to control the larva-to-adult transition

Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.

MiRNAs give worms the time of their lives: Small RNAs and temporal control in Caenorhabditis elegans

Alteration in the timing of particular developmental events can lead to major morphological changes that have profound effects on the life history of an organism. Insights into developmental timing mechanisms have been revealed in the model organism Caenorhabditis elegans, in which a regulatory network of heterochronic genes times events during larval development, ensuring that stage-specific programs occur in the appropriate sequence and on schedule. Developmental timing studies in C. elegans led to the landmark discovery of miRNAs and continue to enhance our understanding of the regulation and activity of these small regulatory molecules. Current views of the heterochronic gene pathway are summarized here, with a focus on the ways in which miRNAs contribute to temporal control and how miRNAs themselves are regulated. Finally, the conservation of heterochronic genes and their functions in timing, as well as their related roles in stem cells and cancer, are highlighted.

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.

Heterochronic genes

What does the term heterochronic mean? Heterochronic refers to the development of cells or tissues at an abnormal time relative to other unaffected events in an organism; the latter can thus serve as temporal landmarks. Mutations in heterochronic genes cause certain cells to adopt cell fates normally associated with earlier or later times in development. Heterochronic genes therefore regulate the timing and sequence of developmental events in specific cell lineages, and thereby coordinate events throughout a developing organism. Why study heterochronic genes in worms? The nematode Caenorhabditis elegans provides a particularly tractable model for studying heterochronic genes, owing to its relatively simple and invariant cell lineages. In the four larval stages of its post-embryonic development, C. elegans cells exhibit stage-specific patterns of developmental events, such as cell division and cell fate specification, with each stage separated by a molt. Importantly, neither larval growth nor progression through the molting cycle in the worm are affected by known heterochronic mutations, allowing developmental events to be monitored relative to these temporal landmarks. So, is this just a strange worm phenomenon? No. Heterochronic genes have been reported in many different organisms, including Drosophila (hunchback, Krüppel, pdm, castor) and several species of plants, including Arabidopsis (HASTY) and rice (mori1). What do heterochronic genes do in worms? One class of heterochronic mutations results in an early, or precocious, phenotype in which many of the normal cell fate decisions of a specific larval stage are skipped and much of the worm instead executes the developmental program of a later stage. Examples include mutations in lin-14, lin-28 and lin-41. Worms with such mutations express adult cell fates when the animal is still sexually immature (Figure 1). Conversely, a second class of mutations results in a delayed, or retarded, phenotype in which many normal cell fate decisions are reiterated in subsequent larval stages. Examples include mutations in lin-4 and lin-29. Worms with such mutations do not express certain adult cell fates, despite being sexually mature (Figure 1). Is there conservation in heterochronic gene pathways? An important regulator of the larval-to-adult transition in worms, let-7, is conserved across diverse phyla, having been identified in flies, sea urchins, humans and many other organisms. In addition, it has recently been demonstrated that the C. elegans heterochronic gene lin-57 is a worm homolog of the Drosophila gene hunchback (hence its new name hbl-1). In the developing central nervous system of flies, hunchback regulates the temporal patterning of cell fate specification for neuroblasts. But doesn't hunchback regulate spatial patterning in flies?Hunchback is indeed best known for its role in anterior–posterior patterning in the early fly embryo; it encodes a zinc-finger transcription factor that acts to establish spatial domains of gene expression. Although downstream effectors are unknown in worms, it is possible that hbl-1 is able to establish analogous temporal domains of gene expression. What's the deal with microRNAs and how are they involved in the heterochronic gene pathway? microRNAs are now appreciated as a new class of small non-coding RNAs. They are likely to act by regulating translation of target mRNAs through binding to sequences in the 3′ untranslated regions with partial anti-sense complementarity. When the product of the lin-4 heterochronic gene was found to be a small, non-coding RNA in 1993, it appeared that it might be a C. elegans-specific, molecular oddity. A second small non-coding RNA, let-7, was discovered in the heterochronic gene pathway in 2000, followed by the cloning of hundreds of small non-coding RNAs (microRNAs) in 2001 and beyond, and it is now appreciated that the heterochronic genes lin-4 and let-7 are the founding members of a larger microRNA gene family. Are microRNAs involved in other pathways? Although the first two microRNAs identified are in the heterochronic gene pathway, there is now evidence for roles of microRNAs in cell proliferation, cell death, fat metabolism and stress resistance in flies. Is there anything we don't know? The mechanism of action of many heterochronic genes remains to be determined. For example, lin-14 encodes a novel nuclear protein with no identified downstream effectors to date. In addition, emerging evidence of phylogenetic conservation of heterochronic genes leads to the prospect that mechanisms to control developmental timing will be elucidated in other systems. Where can I find out more?

The Caenorhabditis elegans hunchback-like Gene lin-57/hbl-1 Controls Developmental Time and Is Regulated by MicroRNAs

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback ( hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3′UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3′UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.

The temporal control of cell cycle and cell fate in Caenorhabditis elegans.

The nematode Caenorhabditis elegans develops through two major phases: the first phase, embryogenesis, consists of a rapid series of cleavage cell divisions leading to morphogenesis of a first stage larva. The second phase is postembryonic development, which consists of developmentally regulated cell cycles that occur during the four larval stages leading to the adult. Precursor cells set aside during embryogenesis divide through stereotypical cell lineage patterns during the four larval stages to generate larval and adult structures. The precise timing of the postembryonic cell divisions is under strict control, in most cases with a developmentally regulated G1. In certain postembryonic cell lineages, various aspects of the cell division cycle, including cell cycle exit, or G1/S progression, are controlled by temporal regulatory genes of the heterochronic gene pathway. Heterochronic genes also control the timing of numerous other developmental events, indicating that this pathway functions to coordinate the schedule of cell division and cellular differentiation throughout the animal. Some choices of cell fate that occur in response to inductive or lateral signals are linked to cell cycle progression, suggesting that cell cycle phase can confer a critical period for developmental potential in certain cells.

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans.

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.

Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene.

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.

Stage-specific accumulation of the terminal differentiation factor LIN-29 during Caenorhabditis elegans development.

The Caenorhabditis elegans gene lin-29 is required for the terminal differentiation of the lateral hypodermal seam cells during the larval-to-adult molt. We find that lin-29 protein accumulates in the nuclei of these cells, consistent with its predicted role as a zinc finger transcription factor. The earliest detectable LIN-29 accumulation in seam cell nuclei is during the last larval stage (L4), following the final seam cell division, which occurs during the L3-to-L4 molt. LIN-29 accumulates in all hypodermal nuclei during the L4 stage. The time of LIN-29 appearance in the hypodermis is controlled by the heterochronic gene pathway: LIN-29 accumulates in the hypodermis abnormally early, during the third larval stage, in loss-of-function lin-14, lin-28 and lin-42 mutants, and fails to accumulate in hypodermis of lin-4 mutants. LIN-29 also accumulates stage-specifically in the nuclei of a variety of non-hypodermal cells during development. Its accumulation is dependent upon the upstream heterochronic genes in some, but not all, of these non-hypodermal cells.

The Caenorhabditis elegans heterochronic gene pathway controls stage-specific transcription of collagen genes.

In Caenorhabditis elegans, the terminal differentiation of the hypodermal cells occurs at the larval-to-adult molt, and is characterized in part by the formation of a morphologically distinct adult cuticle. The timing of this event is controlled by a pathway of heterochronic genes that includes the relatively direct regulatory gene, lin-29, and upstream genes lin-4, lin-14 and lin-28. Using northern analysis to detect endogenous collagen mRNA levels and collagen/lacZ reporter constructs to monitor collagen transcriptional activity, we show that the stage-specific switch from larval cuticle to adult cuticle correlates with the transcriptional activation of adult-specific collagen genes and repression of larval-specific collagen genes. Heterochronic mutations that cause precocious formation of adult cuticle also cause precocious transcription of the adult-specific collagen genes, col-7 and col-19; heterochronic mutations that prevent the switch to adult cuticle cause continued expression of the larval collagen gene, col-17, in adults and prevent adult-specific activation of col-7 or col-19. A 235 bp segment of col-19 5' sequences is sufficient to direct the adult-specific expression of a col-19/lacZ reporter gene in hypodermal cells. These findings indicate that the heterochronic gene pathway regulates the timing of hypodermal cell terminal differentiation by regulating larval- and adult-specific gene expression, perhaps by the direct action of lin-29.