Bruce Wightman

The 20 years it took to recognize the importance of tiny RNAs

Our interest in the C. elegans heterochronic genes began during Gary Ruvkuns post-PhD thesis defense seminar tour of Europe in November, 1981. He took along a Xerox of one paper, a 1981 Cell paper by Marty Chalfie, Bob Horvitz, and John Sulston, describing the detailed cell lineage analysis, but not the molecular identity, of two genes that affect C. elegans developmental timing, lin-4 and unc-86 (Chalfie et al., 1981). The paper was replete with specialized language and concepts that he could not decipher; his bacterial genetics training did not prepare him for the patois of C. elegans developmental genetics, a product of the island tribe that evolved around Sydney Brenner at the Medical Research Council labs in Cambridge, England. Ruvkun visited the MRC on that trip and spent a few hours talking with Marty Chalfie about lin-4 and unc-86. That one afternoon at the MRC planted a seed: Ruvkun glimpsed the worm community, its ambition, its exuberance, its collaborative reflexes, its sense of mission. And the field seemed ready to explode at that moment-there were lots of interesting mutants that were a few technical developments away from exciting molecular discovery. The attraction of C. elegans developmental genetics reasserted itself after Bob Horvitz gave a departmental seminar at Harvard in January of 1982 that was just as confusing and interesting to Ruvkun as the 1981 Cell paper. He went to MIT to talk about worms with Horvitz. Horvitz was very enthusiastic about cracking the problem of going molecular with these very promising genes identified by their genetics. Meeting with Horvitz was Ruvkuns second glimpse of the MRC worm culture, now transplanted to MIT by Horvitz, where it also transmuted to include a sense of urgency. Ruvkun began to work on the problem, part time in Wally Gilberts lab, where he had begun his postdoctoral work and which was a center of molecular biology expertise, and part time in the Horvitz lab, where he would learn worm genetics. Victor Ambros had just finished his genetic analysis of heterochronic genes (Ambros and Horvitz, 1984). The most compelling of the genes was lin-14, because it had both gain-of-function and loss-of-function mutant alleles with opposite developmental timing defects. Such genetic attributes define switch genes, which were

A Transparent Window into Biology: A Primer on Caenorhabditis elegans

A little over 50 years ago, Sydney Brenner had the foresight to develop the nematode (round worm) Caenorhabditis elegans as a genetic model for understanding questions of developmental biology and neurobiology. Over time, research on C. elegans has expanded to explore a wealth of diverse areas in modern biology including studies of the basic functions and interactions of eukaryotic cells, host–parasite interactions, and evolution. C. elegans has also become an important organism in which to study processes that go awry in human diseases. This primer introduces the organism and the many features that make it an outstanding experimental system, including its small size, rapid life cycle, transparency, and well-annotated genome. We survey the basic anatomical features, common technical approaches, and important discoveries in C. elegans research. Key to studying C. elegans has been the ability to address biological problems genetically, using both forward and reverse genetics, both at the level of the entire organism and at the level of the single, identified cell. These possibilities make C. elegans useful not only in research laboratories, but also in the classroom where it can be used to excite students who actually can see what is happening inside live cells and tissues.

A Transparent window into biology: A primer on Caenorhabditis elegans.

A little over 50 years ago, Sydney Brenner had the foresight to develop the nematode (round worm) Caenorhabditis elegans as a genetic model for understanding questions of developmental biology and neurobiology. Over time, research on C. elegans has expanded to explore a wealth of diverse areas in modern biology including studies of the basic functions and interactions of eukaryotic cells, host-parasite interactions, and evolution. C. elegans has also become an important organism in which to study processes that go awry in human diseases. This primer introduces the organism and the many features that make it an outstanding experimental system, including its small size, rapid life cycle, transparency, and well-annotated genome. We survey the basic anatomical features, common technical approaches, and important discoveries in C. elegans research. Key to studying C. elegans has been the ability to address biological problems genetically, using both forward and reverse genetics, both at the level of the entire organism and at the level of the single, identified cell. These possibilities make C. elegans useful not only in research laboratories, but also in the classroom where it can be used to excite students who actually can see what is happening inside live cells and tissues.

The Short Coiled-Coil Domain-Containing Protein UNC-69 Cooperates with UNC-76 to Regulate Axonal Outgrowth and Normal Presynaptic Organization in Caenorhabditis elegans

Background The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however. Results We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects. Conclusion We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly.

The tailless ortholog nhr-67 functions in the development of the C. elegans ventral uterus

The development of the C. elegans uterus provides a model for understanding the regulatory pathways that control organogenesis. In C. elegans, the ventral uterus develops through coordinated signaling between the uterine anchor cell (AC) and a ventral uterine (VU) cell. The nhr-67 gene encodes the nematode ortholog of the tailless nuclear receptor gene. Fly and vertebrate tailless genes function in neuronal and ectodermal developmental pathways. We show that nhr-67 functions in multiple steps in the development of the C. elegans uterus. First, it functions in the differentiation of the AC. Second, it functions in reciprocal signaling between the AC and an equipotent VU cell. Third, it is required for a later signaling event between the AC and VU descendants. nhr-67 is required for the expression of both the lag-2/Delta signal in the AC and the lin-12/Notch receptor in all three VU cells and their descendants, suggesting that nhr-67 may be a key regulator of Notch-signaling components. We discuss the implications of these findings for proposed developmental regulatory pathways that include the helix-loop-helix regulator hlh-2/daughterless and transcription factor egl-43/Evi1 in the differentiation of ventral uterine cell types.

Integration of Bioinformatics into an Undergraduate Biology Curriculum and the Impact on Development of Mathematical Skills.

The development of fields such as bioinformatics and genomics has created new challenges and opportunities for undergraduate biology curricula. Students preparing for careers in science, technology, and medicine need more intensive study of bioinformatics and more sophisticated training in the mathematics on which this field is based. In this study, we deliberately integrated bioinformatics instruction at multiple course levels into an existing biology curriculum. Students in an introductory biology course, intermediate lab courses, and advanced project‐oriented courses all participated in new course components designed to sequentially introduce bioinformatics skills and knowledge, as well as computational approaches that are common to many bioinformatics applications. In each course, bioinformatics learning was embedded in an existing disciplinary instructional sequence, as opposed to having a single course where all bioinformatics learning occurs. We designed direct and indirect assessment tools to follow student progress through the course sequence. Our data show significant gains in both student confidence and ability in bioinformatics during individual courses and as course level increases. Despite evidence of substantial student learning in both bioinformatics and mathematics, students were skeptical about the link between learning bioinformatics and learning mathematics. While our approach resulted in substantial learning gains, student “buy‐in” and engagement might be better in longer project‐based activities that demand application of skills to research problems. Nevertheless, in situations where a concentrated focus on project‐oriented bioinformatics is not possible or desirable, our approach of integrating multiple smaller components into an existing curriculum provides an alternative.

Analysis of C. elegans NR2E nuclear receptors defines three conserved clades and ligand-independent functions

BackgroundThe nuclear receptors (NRs) are an important class of transcription factors that are conserved across animal phyla. Canonical NRs consist of a DNA-binding domain (DBD) and ligand-binding domain (LBD). While most animals have 20–40 NRs, nematodes of the genus Caenorhabditis have experienced a spectacular proliferation and divergence of NR genes. The LBDs of evolutionarily-conserved Caenorhabditis NRs have diverged sharply from their Drosophila and vertebrate orthologs, while the DBDs have been strongly conserved. The NR2E family of NRs play critical roles in development, especially in the nervous system. In this study, we explore the phylogenetics and function of the NR2E family of Caenorhabditis elegans, using an in vivo assay to test LBD function.ResultsPhylogenetic analysis reveals that the NR2E family of NRs consists of three broadly-conserved clades of orthologous NRs. In C. elegans, these clades are defined by nhr-67, fax-1 and nhr-239. The vertebrate orthologs of nhr-67 and fax-1 are Tlx and PNR, respectively. While the nhr-239 clade includes orthologs in insects (Hr83), an echinoderm, and a hemichordate, the gene appears to have been lost from vertebrate lineages. The C. elegans and C. briggsae nhr-239 genes have an apparently-truncated and highly-diverged LBD region. An additional C. elegans NR2E gene, nhr-111, appears to be a recently-evolved paralog of fax-1; it is present in C. elegans, but not C. briggsae or other animals with completely-sequenced genomes. Analysis of the relatively unstudied nhr-111 and nhr-239 genes demonstrates that they are both expressed—nhr-111 very broadly and nhr-239 in a small subset of neurons. Analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions.ConclusionsOur analysis supports three conserved clades of NR2E receptors, only two of which are represented in vertebrates, indicating three ancestral NR2E genes in the urbilateria. The lack of a requirement for a FAX-1 LBD suggests that the relatively high level of sequence divergence for Caenorhabditis LBDs reflects relaxed selection on the primary sequence as opposed to divergent positive selection. This observation is consistent with a model in which divergence of some Caenorhabditis LBDs is allowed, at least in part, by the absence of a ligand requirement.

Conserved and Exapted Functions of Nuclear Receptors in Animal Development

The nuclear receptor gene family includes 18 members that are broadly conserved among multiple disparate animal phyla, indicating that they trace their evolutionary origins to the time at which animal life arose. Typical nuclear receptors contain two major domains: a DNA-binding domain and a C-terminal domain that may bind a lipophilic hormone. Many of these nuclear receptors play varied roles in animal development, including coordination of life cycle events and cellular differentiation. The well-studied genetic model systems of Drosophila, C. elegans, and mouse permit an evaluation of the extent to which nuclear receptor function in development is conserved or exapted (repurposed) over animal evolution. While there are some specific examples of conserved functions and pathways, there are many clear examples of exaptation. Overall, the evolutionary theme of exaptation appears to be favored over strict functional conservation. Despite strong conservation of DNA-binding domain sequences and activity, the nuclear receptors prove to be highly-flexible regulators of animal development.