Patrick Callaerts

Cephalopod Hox genes and the origin of morphological novelties

Cephalopods are a diverse group of highly derived molluscs, including nautiluses, squids, octopuses and cuttlefish. Evolution of the cephalopod body plan from a monoplacophoran-like ancestor entailed the origin of several key morphological innovations contributing to their impressive evolutionary success. Recruitment of regulatory genes, or even pre-existing regulatory networks, may be a common genetic mechanism for generating new structures. Hox genes encode a family of transcriptional regulatory proteins with a highly conserved role in axial patterning in bilaterians; however, examples highlighting the importance of Hox gene recruitment for new developmental functions are also known. Here we examined developmental expression patterns for eight out of nine Hox genes in the Hawaiian bobtail squid Euprymna scolopes, by whole-mount in situ hybridization. Our data show that Hox orthologues have been recruited multiple times and in many ways in the origin of new cephalopod structures. The manner in which these genes have been co-opted during cephalopod evolution provides insight to the nature of the molecular mechanisms driving morphological change in the Lophotrochozoa, a clade exhibiting the greatest diversity of body plans in the Metazoa.

Drosophila Pax-6/eyeless is essential for normal adult brain structure and function.

A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed.

Automated protein-DNA interaction screening of Drosophila regulatory elements

Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation, we need convenient tools to identify the proteins that bind to them. Here we describe the development and validation of a high-throughput yeast one-hybrid platform, which enables screening of DNA elements versus an array of full-length, sequence-verified clones containing over 85% of predicted Drosophila transcription factors. Using six well-characterized regulatory elements, we identified 33 transcription factor–DNA interactions of which 27 were previously unidentified. To simultaneously validate these interactions and locate the binding sites of involved transcription factors, we implemented a powerful microfluidics-based approach that enabled us to retrieve DNA-occupancy data for each transcription factor throughout the respective target DNA elements. Finally, we biologically validated several interactions and identified two new regulators of sine oculis gene expression and hence eye development.

miRNA genes and the brain: implications for psychiatric disorders.

MicroRNAs (miRNAs) are a class of nonprotein coding genes with a growing importance in regulatory mechanisms of gene expression related to brain function and plasticity. Considering the relative lack of success of the analysis of variations in candidate protein coding genes and of genome‐wide association studies to identify strong risk factors for common psychiatric disorders (PDs), miRNA genes are of particular interest for the field of psychiatric genetics as deregulation of the rate of transcription or translation of a normal gene may be phenotypically similar to disruption of the gene itself. In this article we review the current knowledge on the contribution of miRNAs in basic mechanisms of brain development and plasticity and their possible involvement in the pathogenesis of several PDs. Because future functional and genomic explorations of brain expressed miRNAs, and other types of noncoding RNAs, may identify additional candidate genes and pathways for common PDs, we believe that implementing additional strategies to further elucidate the role of miRNAs in the etiology of common PDs is of great importance. Hum Mutat 31:1–10, 2010.

Characterization and expression analysis of an ancestor-type Pax gene in the hydrozoan jellyfish Podocoryne carnea

We characterized a Pax gene from the hydrozoan Podocoryne carnea. It is most similar to cnidarian Pax-B genes and encodes a paired domain, a homeodomain and an octapeptide. Expression analysis demonstrates the presence of Pax-B transcripts in eggs, the ectoderm of the planula larva and in a few scattered cells in the apical polyp ectoderm. In developing and mature medusae, Pax-B is localized in particular endodermal cells, oriented toward the outside. Pax-B is not expressed in muscle cells. However, if isolated striated muscle tissue is activated for transdifferentiation, the gene is expressed within 1 h, before new cell types, such as smooth muscle and nerve cells, have formed. The expression data indicate that Pax-B is involved in nerve cell differentiation.

Gene duplication and recruitment of a specific tropomyosin into striated muscle cells in the jellyfish Podocoryne carnea

Cnidaria are the most basal animal phylum in which smooth and striated muscle cells have evolved. Since the ultrastructure of the mononucleated striated muscle is similar to that of higher animals, it is of interest to compare the striated muscle of Cnidaria at the molecular level to that of triploblastic phyla. We have used tropomyosins, a family of actin binding proteins to address this question. Throughout the animal kingdom, a great diversity of tropomyosin isoforms is found in non-muscle cells but only a few conserved tropomyosins are expressed in muscle cells. Muscle tropomyosins are all similar in length and share conserved termini. Two cnidarian tropomyosins have been described previously but neither of them is expressed in striated muscle cells. Here, we have characterized a new tropomyosin gene Tpm2 from the hydrozoan Podocoryne carnea. Expression analysis by RT-PCR and by whole mount in situ hybridization demonstrate that Tpm2 is exclusively expressed in striated muscle cells of the medusa. The Tpm2 protein is shorter in length than its counterparts from higher animals and differs at both amino and carboxy termini from striated muscle isoforms of higher animals. Interestingly, Tpm2 differs considerably from Tpm1 (only 19% identity) which was described previously in Podocoryne carnea. This divergence indicates a functional separation of cytoskeletal and striated muscle tropomyosins in cnidarians. These data contribute to our understanding of the evolution of the tropomyosin gene family and demonstrate the recruitment of tropomyosin into hydrozoan striated muscles during metazoan evolution. J. Exp. Zool. (Mol. Dev. Evol.) 285:378-386, 1999.

The Hawaiian bobtail squid (Euprymna scolopes): a model to study the molecular basis of eukaryote-prokaryote mutualism and the development and evolution of morphological novelties in cephalopods.

The Hawaiian bobtail squid, Euprymna scolopes, is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. E. scolopes is studied in three principal contexts: (1) as a model of cephalopod development; (2) as a model of animal-bacterial symbioses; and (3) as a system for studying adaptations of tissues that interact with light. E. scolopes embryos can be obtained continually and can be reared in the laboratory over an entire generation. The embryos and protective chorions are optically clear, facilitating in situ developmental observations, and can be manipulated experimentally. Many molecular protocols have been developed for studying E. scolopes development. This species is best known, however, for its symbiosis with the luminous marine bacterium Vibrio fischeri and has been used to study determinants of symbiont specificity, the influence of symbiosis on development of the squid light organ, and the mechanisms by which a stable association is achieved. Both partners can be grown independently under laboratory conditions, a feature that offers the unusual opportunity to manipulate the symbiosis experimentally. Molecular and genetic tools have been developed for V. fischeri, and a large expressed sequence tag (EST) database is available for the host symbiotic tissues. Additionally, comparisons between light organ form and function to those of the eye can be made. Both types of tissue interact with light, but have divergent embryonic development. As such, they offer an opportunity to study the molecular basis for the evolution of morphological novelties.

Mutational analysis of the eyeless gene and phenotypic rescue reveal that an intact Eyeless protein is necessary for normal eye and brain development in Drosophila

Pax6 genes encode evolutionarily highly conserved transcription factors that are required for eye and brain development. Despite the characterization of mutations in Pax6 homologs in a range of organisms, and despite functional studies, it remains unclear what the relative importance is of the various parts of the Pax6 protein. To address this, we have studied the Drosophila Pax6 homolog eyeless. Specifically, we have generated new eyeless alleles, each with single missense mutations in one of the four domains of the protein. We show that these alleles result in abnormal eye and brain development while maintaining the OK107 eyeless GAL4 activity from which they were derived. We performed in vivo functional rescue experiments by expressing in an eyeless-specific pattern Eyeless proteins in which either the paired domain, the homeodomain, or the C-terminal domain was deleted. Rescue of the eye and brain phenotypes was only observed when full-length Eyeless was expressed, while all deletion constructs failed to rescue. These data, along with the phenotypes observed in the four newly characterized eyeless alleles, demonstrate the requirement for an intact Eyeless protein for normal Drosophila eye and brain development. They also suggest that some endogenous functions may be obscured in ectopic expression experiments.

A network of synaptic genes associated with schizophrenia and bipolar disorder

Identification of novel candidate genes for schizophrenia (SZ) and bipolar disorder (BP), two psychiatric disorders with large epidemiological impacts, is a key research area in neurosciences and psychiatric genetics. Previous evidence from genome-wide studies suggests an important role for genes involved in synaptic plasticity in the risk for SZ and BP. We used a convergent genomics approach, combining different lines of biological evidence, to identify genes involved in the cAMP/PKA/CREB functional pathway that could be novel candidates for BP and SZ: CREB1, CREM, GRIN2C, NPY2R, NF1, PPP3CB and PRKAR1A. These 7 genes were analyzed in a HapMap based association study comprising 48 common SNPs in 486 SZ, 351 BP patients and 514 control individuals recruited from an isolated population in Northern Sweden. Genetic analysis showed significant allelic associations of SNPs in PRKAR1A with SZ and of PPP3CB and PRKAR1A with BP. Our results highlight the feasibility and the importance of convergent genomic data analysis for the identification of candidate genes and our data provide support for the role of common inherited variants in synaptic genes and their involvement in the etiology of BP and SZ.

Whole-mount in situ hybridization of Hawaiian bobtail squid (Euprymna scolopes) embryos with DIG-labeled riboprobes: II. Embryo preparation, hybridization, washes, and immunohistochemistry.

Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody.