Control of developmental timing in animals.

The molecular mechanisms that time development are now being deciphered in various organisms, particularly in Caenorhabditis elegans. Key recent findings indicate that certain C. elegans timekeeping genes are conserved across phyla, and their developmental expression patterns indicate that a timing function might also be conserved. Small regulatory RNAs have crucial roles in the timing mechanism, and the cellular machinery required for production of these RNAs intersects with that used to process double-stranded RNAs during RNA interference.

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.

Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins.

The Caenorhabditis elegans heterochronic genes control the relative timing and sequence of many events during postembryonic development, including the terminal differentiation of the lateral hypodermis, which occurs during the final (fourth) molt. Inactivation of the heterochronic gene lin-42 causes hypodermal terminal differentiation to occur precociously, during the third molt. LIN-42 most closely resembles the Period family of proteins from Drosophila and other organisms, proteins that function in another type of biological timing mechanism: the timing of circadian rhythms. Per mRNA levels oscillate with an approximately 24-hour periodicity. lin-42 mRNA levels also oscillate, but with a faster rhythm; the oscillation occurs relative to the approximately 6-hour molting cycles of postembryonic development.

The LIN-29 transcription factor is required for proper morphogenesis of the Caenorhabditis elegans male tail.

The Caenorhabditis elegans gene lin-29 encodes a zinc-finger transcription factor that is required for hypodermal cell terminal differentiation and proper vulva morphogenesis. Here we demonstrate that lin-29 is also required in males for productive mating. We show that lin-29 males can perform the early mating behaviors including response to hermaphrodite contact and vulva location, but they do not perform the subsequent steps of vulva attachment via spicule insertion and sperm transfer. Consistent with this observation, we found that lin-29 mutant spicules are on average 43% shorter than wild-type spicules while other male mating structures appear unaltered. In lin-29 mutants, spicule development goes awry after the generation of spicule cells, when spicule morphogenesis occurs in wild-type males. We show that LIN-29 accumulates in many cells of the wild-type male tail, including those that form the spicules. We demonstrate, through analysis of genetic mosaics, that the formation of wild-type-length spicules requires lin-29(+) in the AB.p lineage, the lineage that gives rise to the spicules and other male copulatory structures. Our mosaic analysis also reveals a role for lin-29(+) in the P1 lineage, which mainly produces sex muscles, cells of the somatic gonad, and body wall muscles.

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.

The terminal differentiation factor LIN-29 is required for proper vulval morphogenesis and egg laying in Caenorhabditis elegans.

Caenorhabditis elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. We demonstrate through analysis of genetic mosaics that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis. First, LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying. Second, animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis. Finally, vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells.

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 heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans.

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.

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.

Postinitiation transcriptional control in Drosophila melanogaster.

Drosophila hsp70 genes have an RNA polymerase II molecule paused at their 5' ends in uninduced cells. In this study we have shown that this pausing also occurs on other heat shock and constitutively expressed genes. We propose that a rate-limiting step in early elongation occurs in many Drosophila genes and may be a target for transcriptional regulation.

The RNA polymerase II molecule at the 5' end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged.

Protein-DNA cross-linking of cultured Drosophila cells has shown that, in vivo, prior to the induction of heat shock, there is approximately one molecule of RNA polymerase II associated with the promoter region of the major heat shock gene, hsp70. Here, we show that this promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain of approximately 25 nucleotides in length, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction. The detection of a post-initiation RNA polymerase complex on the promoter region of the inactive gene suggests that there is a transcriptional control mechanism that acts at a step early in transcript elongation.

Glutamate dehydrogenase catalyzes the reduction of a Schiff base (delta 1-pyrroline-2-carboxylic acid) by NADPH.

alpha-Iminoglutarate has long been postulated as an obligatory intermediate in the glutamate dehydrogenase catalyzed reaction, but direct proof of its participation is lacking. We report here the glutamate dehydrogenase catalyzed reduction of delta 1-pyrroline-2-carboxylic acid (a cyclic-alpha-imino acid) to proline (an alpha-amino acid). The catalysis occurs at the normal catalytic site of the enzyme. The imine and the enzyme-NADPH complex are the active oxidant and reductant, respectively. The latter is about 500 times more reactive than NADPH itself. These findings provide direct evidence that the glutamate dehydrogenase catalyzed reaction does indeed proceed by way of an enzyme-bound form of an alpha-iminocarboxylic acid.