TCF/Lef regulates the Gsx ParaHox gene in central nervous system development in chordates.

BACKGROUND: The ParaHox genes play an integral role in the anterior-posterior (A-P) patterning of the nervous system and gut of most animals. The ParaHox cluster is an ideal system in which to study the evolution and regulation of developmental genes and gene clusters, as it displays similar regulatory phenomena to its sister cluster, the Hox cluster, but offers a much simpler system with only three genes. RESULTS: Using Ciona intestinalis transgenics, we isolated a regulatory element upstream of Branchiostoma floridae Gsx that drives expression within the central nervous system of Ciona embryos. The minimal amphioxus enhancer region required to drive CNS expression has been identified, along with surrounding sequence that increases the efficiency of reporter expression throughout the Ciona CNS. TCF/Lef binding sites were identified and mutagenized and found to be required to drive the CNS expression. Also, individual contributions of TCF/Lef sites varied across the regulatory region, revealing a partial division of function across the Bf-Gsx-Up regulatory element. Finally, when all TCF/Lef binding sites are mutated CNS expression is not only abolished, but a latent repressive function is also unmasked. CONCLUSIONS: We have identified a B. floridae Gsx upstream regulatory element that drives CNS expression within transgenic Ciona intestinalis, and have shown that this CNS expression is dependent upon TCF/Lef binding sites. We examine the evolutionary and developmental implications of these results, and discuss the possibility of TCF/Lef not only as a regulator of chordate Gsx, but as a deeply conserved regulatory factor controlling all three ParaHox genes across the Metazoa.

Chordate Hox and ParaHox gene clusters differ dramatically in their repetitive element content.

The ParaHox and Hox gene clusters control aspects of animal anterior-posterior development and are related as paralogous evolutionary sisters. Despite this relationship, it is not clear if the clusters operate in similar ways, with similar constraints. To compare clusters, we examined the transposable-element (TE) content of amphioxus and mammalian ParaHox and Hox clusters. Chordate Hox clusters are known to be largely devoid of TEs, possibly due to gene regulation and constraints on clustering in these animals. Here, we describe several novel amphioxus TEs and show that the amphioxus ParaHox cluster is a hotspot for TE insertion. TE contents of mammalian ParaHox loci are at background levels, in stark contrast to chordate Hox clusters. This marks a significant difference between Hox and ParaHox clusters. The presence of so many potentially disruptive elements implies selection constrains these ParaHox clusters as they have not dispersed despite 500 My of evolution for each lineage.

Retinoic acid and Wnt/beta-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus.

A role for Wnt/beta-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/beta-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/beta-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/beta-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/beta-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo-increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.

Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae).

The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid (RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posterior position. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes. By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster.

Patterns of conservation and change in honey bee developmental genes.

The current insect genome sequencing projects provide an opportunity to extend studies of the evolution of developmental genes and pathways in insects. In this paper we examine the conservation and divergence of genes and developmental processes between Drosophila and the honey bee; two holometabolous insects whose lineages separated approximately 300 million years ago, by comparing the presence or absence of 308 Drosophila developmental genes in the honey bee. Through examination of the presence or absence of genes involved in conserved pathways (cell signaling, axis formation, segmentation and homeobox transcription factors), we find that the vast majority of genes are conserved. Some genes involved in these processes are, however, missing in the honey bee. We have also examined the orthology of Drosophila genes involved in processes that differ between the honey bee and Drosophila. Many of these genes are preserved in the honey bee despite the process in which they act in Drosophila being different or absent in the honey bee. Many of the missing genes in both situations appear to have arisen recently in the Drosophila lineage, have single known functions in Drosophila, and act early in developmental pathways, while those that are preserved have pleiotropic functions. An evolutionary interpretation of these data is that either genes with multiple functions in a common ancestor are more likely to be preserved in both insect lineages, or genes that are preserved throughout evolution are more likely to co-opt additional functions.

Expression of Pax group III genes in the honeybee (Apis mellifera).

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior-posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.