Of blades and branches: understanding and expanding the Arabidopsis ad/abaxial regulatory network through target gene identification.

HD-ZIPIII and KANADI transcription factors have opposing and dramatic affects on plant development. Analysis of mutants shows these proteins to be master regulators of ad/abaxial (i.e., upper/lower) leaf polarity, leaf blade outgrowth, and branch formation. Because these factors do their work by regulating other genes, we have focused our attention on defining their targets. We have found overlap between the ad/abaxial regulatory pathway and hormone signaling pathways, especially pathways of abscisic acid and auxin signaling. This has led to the discovery that abscisic acid signaling acts upstream of HD-ZIPIII and KANADI in the control of germination and may ultimately explain how environmental stress pathways control new growth at the shoot apex. Auxin signaling conversely is downstream from HD-ZIPIII and KANADI action with these factors controlling targets at all steps of auxin action-biosynthesis, transport, regulation of transport, and signaling. Based on these findings, we propose a model in which the HD-ZIPIII and KANADI factors pattern auxin response in the embryo. Finally, many genes targeted for control by HD-ZIPIII and KANADI proteins are themselves transcription factors-indicating these master regulators call up tissue specific subprograms of transcriptional control to affect the many polar differences observed across tissues.

Isoform-specific mutations in the Caenorhabditis elegans heterochronic gene lin-14 affect stage-specific patterning.

The Caenorhabditis elegans heterochronic gene lin-14 specifies the temporal sequence of postembryonic developmental events. lin-14, which encodes differentially spliced LIN-14A and LIN-14B1/B2 protein isoforms, acts at distinct times during the first larval stage to specify first and second larval stage-specific cell lineages. Proposed models for the molecular basis of these two lin-14 gene activities have included the production of functionally distinct isoforms and the generation of a temporal gradient of LIN-14 protein. We report here that loss of the LIN-14B1/B2 isoforms alone affects one of the two lin-14 temporal patterning functions, the specification of second larval stage lineages. A temporal expression difference between LIN-14A and LIN-14B1/B2 is not responsible for the stage-specific phenotype: protein levels of all LIN-14 isoforms are high in early first larval stage animals and decrease during the first larval stage. However, LIN-14A can partially substitute for LIN-14B1/B2 when expressed at a higher-than-normal level in the late L1 stage. These data indicate that LIN-14B1/B2 isoforms do not provide a distinct function of the lin-14 locus in developmental timing but rather may contribute to an overall level of LIN-14 protein that is the critical determinant of temporal cell fate.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA.

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

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.

Graded expression of ceh-14 reporters in the hypodermis is induced by a gonadal signal.

ceh-14, a LIM class homeobox gene from Caenorhabditis elegans, is the orthologue of the vertebrate Lhx3/Lhx4 genes. ceh-14 reporter constructs are expressed in several different cell types: head and tail neurons, spermatheca and hypodermis. An intriguing aspect of the hypodermal expression pattern is that it takes the form of a gradient which is strongest in the central body region in L4 to young adult hermaphrodites. Promoter deletion analyses revealed that important regulatory elements for hypodermal expression are located within the transcribed region of ceh-14. Since a large part of the hypodermis is a syncytium, we hypothesized that this expression is triggered in a non-cell-autonomous fashion, a possible source being the underlying gonad. In males, which have a different gonadal organisation, the ceh-14 reporter constructs are expressed in a gradient that is strongest in the tail. By laser ablation of the gonadal precursor cells we found that ceh-14 reporter construct expression is eliminated in the hermaphrodite hypodermis, suggesting that the gonad plays a role in the generation of the gradient. Several signaling pathways are known in the gonad and the vulva, thus we crossed the mutations lin-3, egl-17 and lin-12 with the ceh-14 reporter lines. However, the expression of the reporter constructs is not affected in these mutant backgrounds. This suggests that another, presently unknown, signal triggers the graded hypodermal expression.