Biomedical subjects
Publications and source records attributed to V Ambros.
Animal genomes contain an abundance of small genes that produce regulatory RNAs of about 22 nucleotides in length. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they may participate in a wide range of genetic regulatory pathways.
The lin-4 and let-7 antisense RNAs are temporal regulators that control the timing of developmental events in Caenorhabditis elegans by inhibiting translation of target mRNAs. let-7 RNA is conserved among bilaterian animals, suggesting that this class of small RNAs [microRNAs (miRNAs)] is evolutionarily ancient. Using bioinformatics and cDNA cloning, we found 15 new miRNA genes in C. elegans. Several of these genes express small transcripts that vary in abundance during C. elegans larval development, and three of them have apparent homologs in mammals and/or insects. Small noncoding RNAs of the miRNA class appear to be numerous and diverse.
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.
Studies of the nematode Caenorhabditis elegans have identified genetic and molecular mechanisms controlling temporal patterns of developmental events. Mutations in genes of the C. elegans heterochronic pathway cause altered temporal patterns of larval development, in which cells at certain larval stages execute cell division patterns or differentiation programs normally specific for other stages. The products of the heterochronic genes include transcriptional and translational regulators and two different cases of novel small translational regulatory RNAs. Other genes of the pathway encode evolutionarily conserved proteins, including a homolog of the Drosophila Period circadian timing regulator, and a member of the nuclear receptor family of proteins. These regulators interact with each other to elaborate stage-specific regulatory switches and act through downstream effectors to control the timing of cell-type-specific developmental events.
Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. We suggest that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29.
During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternative lin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternative lin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.
lin-4 encodes a small RNA that is complementary to sequences in the 3' untranslated region (UTR) of lin-14 mRNA and that acts to developmentally repress the accumulation of LIN-14 protein. This repression is essential for the proper timing of numerous events of Caenorhabditis elegans larval development. We have investigated the mechanism of lin-4 RNA action by examining the fate of lin-14 mRNA in vivo during the time that lin-4 RNA is expressed. Our results indicate that the rate of synthesis of lin-14 mRNA, its state of polyadenylation, its abundance in the cytoplasmic fraction, and its polysomal sedimentation profile do not change in response to the accumulation of lin-4 RNA. Our results indicate that association of lin-4 RNA with the 3' UTR of lin-14 mRNA permits normal biogenesis of lin-14 mRNA, and normal translational initiation, but inhibits step(s) thereafter, such as translational elongation and/or the release of stable LIN-14 protein.
The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of lin-4 RNA accumulation corresponded to the timing of LIN-14 protein down-regulation. Transgenic strains that express elevated levels of lin-4 RNA prior to 12 h of development display reduced levels of LIN-14 protein and precocious phenotypes consistent with abnormally early loss of lin-14 activity. These results indicate that the temporal profile of lin-4 RNA accumulation specifies the timing of LIN-14 down-regulation and thereby controls the timing of postembryonic developmental events.
In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees ) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees ) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees ) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two developmental decisions separable by cell cycle phase: lin-12 must act prior to the end of VPC S phase to influence a 1 degrees versus 2 degrees cell fate choice, but must act after VPC S phase to influence a 3 degrees versus 2 degrees cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells.
C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.
Mutations in the heterochronic gene lin-28 of C. elegans cause precocious development where diverse events specific to the second larval stage are skipped. lin-28 encodes a cytoplasmic protein with a cold shock domain and retroviral-type (CCHC) zinc finger motifs, consistent with a role for LIN-28 in posttranscriptional regulation. The 3'UTR of lin-28 contains a conserved element that is complementary to the 22 nt regulatory RNA product of lin-4 and that resembles seven such elements in the 3'UTR of the heterochronic gene lin-14. Both lin-4 activity and the lin-4-complementary element (LCE) are necessary for stage-specific regulation of lin-28. Deleting the LCE produces a dominant gain-of-function allele that causes a retarded phenotype, indicating that lin-28 activity is a switch that controls choices of stage-specific fates.
Heterochronic genes control the timing of vulval development in the C. elegans hermaphrodite. lin-14 or lin-28 loss-of-function mutations cause the vulval precursor cells (VPCs) to enter S phase and to divide one larval stage earlier than in the wild type. A precocious vulva is formed by essentially normal cell lineage patterns, governed by the same intercellular signals as in the wild type. Mutations that prevent the normal developmental down-regulation of lin-14, activity delay or block VPC division and prevent vulval differentiation. A genetic pathway that includes lin-4, lin-14, and lin-28 controls when VPCs complete G1 and also controls when VPCs acquire the competence to respond to the intercellular patterning signals and express vulval fates.
In Caenorhabditis elegans, the fates of the multipotent vulval precursor cells (VPCs) are specified by intercellular signals. The VPCs divide in the third larval stage (L3) of the wild type, producing progeny of determined cell types. In lin-28 mutants, vulva development is similar to wild-type vulva development except that it occurs precociously, in the second larval stage (L2). Consequently, when lin-28 hermaphrodites temporarily arrest development at the end of L2 in the dauer larva stage, they have partially developed vulvae consisting of VPC progeny. During post-dauer development, these otherwise determined VPC progeny become reprogrammed back to the multipotent, signal-sensitive state of VPCs. Our results indicate that VPC fate determination by intercellular signals is reversible by dauer larva developmental arrest and post-dauer development.
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.
A hierarchy of heterochronic genes, lin-4, lin-14, lin-28 and lin-29, temporally restricts terminal differentiation of Caenorhabditis elegans hypodermal seam cells to the final molt. This terminal differentiation event involves cell cycle exit, cell fusion and the differential regulation of genes expressed in the larval versus adult hypodermis. lin-29 is the most downstream gene in the developmental timing pathway and thus it is the most direct known regulator of these diverse processes. We show that lin-29 encodes a protein with five zinc fingers of the (Cys)2-(His)2 class and thus likely controls these processes by regulating transcription in a stage-specific manner. Consistent with this role, a lin-29 fusion protein binds in vitro to the 5' regulatory sequences necessary in vivo for expression of col-19, a collagen gene expressed in the adult hypodermis. lin-29 mRNA is detected in the first larval stage and increases in abundance through subsequent larval stages until the final molt, when lin-29 activity is required for terminal differentiation.
The heterochronic genes of Caenorhabditis elegans encode part of a regulatory system that controls the temporal component of cell fates in development. The genes have been characterized genetically and molecularly, and their study has so far revealed a genetic hierarchy that specifies sequences of developmental events, a novel RNA-mediated mechanism of gene regulation and a reprogramming phenomenon associated with arrested development.
lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans. lin-4 acts by negatively regulating the level of LIN-14 protein, creating a temporal decrease in LIN-14 protein starting in the first larval stage (L1). We have cloned the C. elegans lin-4 locus by chromosomal walking and transformation rescue. We used the C. elegans clone to isolate the gene from three other Caenorhabditis species; all four Caenorhabditis clones functionally rescue the lin-4 null allele of C. elegans. Comparison of the lin-4 genomic sequence from these four species and site-directed mutagenesis of potential open reading frames indicated that lin-4 does not encode a protein. Two small lin-4 transcripts of approximately 22 and 61 nt were identified in C. elegans and found to contain sequences complementary to a repeated sequence element in the 3' untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction.