Ann K. Corsi

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 C. elegans Twist target gene, arg-1, is regulated by distinct E box promoter elements

Proper metazoan mesoderm development requires the function of a basic helix-loop-helix (bHLH) transcription factor, Twist. Twist-containing dimers regulate the expression of target genes by binding to E box promoter elements containing the site CANNTG. In Caenorhabditis elegans, CeTwist functions in a subset of mesodermal cells. Our study focuses on how CeTwist controls the expression of its target gene, arg-1. We find that a 385bp promoter region of arg-1, which contains three different E box elements, is sufficient for maintaining the full CeTwist-dependent expression pattern. Interestingly, the expression of arg-1 in different tissues is regulated distinctly, and each of the three E boxes plays a unique role in the regulation. The first and the third E boxes (E1 and E3) are required for expression in a distinct subset of the mesodermal tissues where arg-1 is normally expressed, and the second E box (E2) is required for expression in the full set of those tissues. The essential role of E2 in arg-1 regulation is correlated with the finding that E2 binds with greater affinity than E1 or E3 to CeTwist dimers. A potential role for additional transcription factors in mesodermal gene regulation is suggested by the discovery of a novel site that is also required for arg-1 expression in a subset of the tissues but is not bound in vitro by CeTwist. On the basis of these results, we propose a model of CeTwist gene regulation in which expression is controlled by tissue-specific binding of distinct sets of E boxes.

C. elegans Twist gene expression in differentiated cell types is controlled by autoregulation through intron elements

The temporospatial regulation of genes encoding transcription factors is important during development. The hlh-8 gene encodes the C. elegans mesodermal transcription factor CeTwist. Elements in the hlh-8 promoter restrict gene expression to predominantly undifferentiated cells of the M lineage. We have discovered that hlh-8 expression in differentiated mesodermal cells is controlled by two well-conserved E box elements in the large first intron. Additionally, we found that these elements are bound in vitro by CeTwist and its transcription factor partner, CeE/DA. The E box driven expression is eliminated or diminished in an hlh-8 null allele or in hlh-2 (CeE/DA) RNAi, respectively. Expression of hlh-8 is also diminished in animals harboring an hlh-8 intron deletion allele. Altogether, our results support a model in which hlh-8 is initially expressed in the undifferentiated M lineage cells via promoter elements and then the CeTwist activates its own expression further (autoregulation) in differentiated cells derived from the M lineage via the intron elements. This model provides a mechanism for how a transcription factor may regulate distinct target genes in cells both before and after initiating the differentiation program. The findings could also be relevant to understanding human Twist gene regulation, which is currently not well understood.

Distinct Caenorhabditis elegans HLH-8/twist-containing dimers function in the mesoderm.

Background: The Caenorhabditis elegans basic helix‐loop‐helix (bHLH) factor HLH‐8, the single Twist ortholog in the nematode genome, plays important roles in mesoderm development, including M lineage patterning and differentiation of vulval and enteric muscles. HLH‐8 cooperates with HLH‐2, the bHLH E/Daughterless ortholog, to regulate downstream target genes, but it is not known whether HLH‐2 is an obligate partner for all HLH‐8 functions. Results: Using hlh‐2 loss‐of‐function alleles and RNAi, we discovered that HLH‐2 is required in the vulval muscles but not in M patterning or enteric muscle development. Additionally, we found that expressing tethered HLH‐8/HLH‐8 dimers in hlh‐8 null animals rescued M patterning and enteric but not vulval muscle development. Conclusions: These results support a model whereby HLH‐8/HLH‐8 homodimers function in M lineage patterning and enteric muscles and HLH‐8/HLH‐2 heterodimers function in the M‐derived vulval muscles. Interestingly, the different dimers function in the same M lineage cells and the switch in dimer function coincides with vulval muscle differentiation. The use of distinct Twist dimers is evolutionarily conserved, and C. elegans provides a paradigm for future dissection of differential promoter regulation by these dimers at a single cell resolution. Developmental Dynamics 241:481–492, 2012.

Books for free? How can this be?— A PubMed resource you may be overlooking

The NCBI (National Center for Biotechnology Information) at the National Institutes of Health collects a wide range of molecular biological data, and develops tools and databases to analyse and disseminate this information. Many life scientists are familiar with the website maintained by the NCBI (http:www.ncbi.nlm.nih.gov), because they use it to search GenBank for homologues of their genes of interest or to search the PubMed database for scientific literature of interest. There is also a database called the Bookshelf that includes searchable popular life science textbooks, medical and research reference books and NCBI reference materials. The Bookshelf can be useful for researchers and educators to find basic biological information. This article includes a representative list of the resources currently available on the Bookshelf, as well as instructions on how to access the information in these resources.

Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.

Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.