The eyes have it: A brief history of crustacean neuroendocrinology.

To help celebrate the 50th anniversary of General and Comparative Endocrinology, the history of only a small portion of crustacean endocrinology is presented here. The field of crustacean endocrinology dates back to the decades prior to the establishment of General and Comparative Endocrinology and the first article about crustacean endocrinology published in this journal was concerned with the anatomy of neurosecretory and neurohemal structures in brachyuran crabs. This review looks at the history of neuroendocrinology in crustaceans during that time and tries to put perspective on the future of this field.

RXR isoforms and endogenous retinoids in the fiddler crab, Uca pugilator.

The pleiotropic effects of circulating ecdysteroids in the adult fiddler crab, Uca pugilator, during molting, regeneration, and reproduction are mediated by a limited number of receptor proteins. We hypothesize that hormonal effects in vivo may be the result of complex interactions between at least two receptor heterodimer conformations that differentially respond to multiple ecdysteroid/retinoid signals. Two splicing variants of the fiddler crab retinoid-X-receptor (UpRXR) differ from one another by the addition of a 33 amino acid insert in the ligand-binding domain. We show here that the ecdysteroid receptor in the fiddler crab (UpEcR) behaves differently depending upon the UpRXR isoform with which it is partnered. The two UpRXR variant partners for UpEcR confer slightly different responses in the binding of Ponasterone A (PA)-a naturally occurring ecdysteroid in the blood of Uca. UpRXR can bind 9-cis retinoic acid (9cRA) as well as terpenoids. 9cRA and the naturally occurring terpenoid, methyl farnesoate, influence the binding of PA to UpEcR/UpRXR dimers. Endogenous retinoids are found in the blastema of regenerating limbs of Uca and they (plus blood-borne terpenoids) may add additional levels of differential response by target tissues. Thus, the two sets of heterodimers tested here may represent different dynamic complexes whose properties are defined by the specific heterodimeric subunits involved and the specific ligands available.

Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor-receptor interaction with a cognate ecdysteroid receptor.

We have identified cDNA clones that encode homologs of the ecdysteroid receptor (EcR) and retinoid-X receptor (RXR)/USP classes of nuclear receptors from the fiddler crab Uca pugilator (UpEcR and UpRXR). Several UpRXR cDNA splicing variants were found in coding regions that could potentially influence function. A five-amino acid (aa) insertion/deletion is located in the "T" box in the hinge region. Another 33-aa insertion/deletion is found inside the ligand-binding domain (LBD), between helix 1 and helix 3. Ribonuclease protection assays (RPA) showed that four UpRXR transcripts [UpRXR(+5+33), UpRXR(-5+33), UpRXR(+5-33) and UpRXR(-5-33)] were present in regenerating limb buds. UpRXR(-5+33) was the most abundant transcript present in regenerating limb buds in both early blastema and late premolt growth stages. Expression vectors for these UpRXR variants and UpEcR were constructed, and the proteins expressed in E. coli and in vitro expression systems. The expressed crab nuclear receptors were then characterized by electrophoretic mobility shift assay (EMSA) and glutathione S-transferase (GST) pull down experiments. EMSA results showed that UpEcR/UpRXR(-5+33) heterocomplexes bound with a series of hormone response elements (HREs) including eip28/29, IRper-1, DR-4, and IRhsp-1 with appreciable affinity. Competition EMSA also showed that the affinity decreased as sequence composition deviated from a perfect consensus element. Binding to IRper-1 HREs occurred only if the heterodimer partner UpRXR contained the 33-aa LBD insertion. UpRXR lacking both the 5-aa and 33-aa insertion bound to a DR-1G HRE in the absence of UpEcR. The results of GST-pull down experiments showed that UpEcR interacted only with UpRXR variants containing the 33-aa insertion, and not with those lacking the 33-aa insertion. These in vitro receptor protein-DNA and receptor protein-protein interactions occurred in the absence of hormone (20-hydroxyecdysone and 9-cis retinoid acid, 9-cis RA). Transactivation studies using a hybrid UpEcR ligand-binding domain construct and UpRXR (+/-33) ligand-binding domain constructs also showed that the 33-aa insertion was indispensable in mediating ecdysteroid stimulated transactivation.

Characterization of crab EcR and RXR homologs and expression during limb regeneration and oocyte maturation.

We report here complete coding sequences for the Uca pugilator homologs of the ecdysteroid (UpEcR) and retinoid-X receptors (UpRXR). Library screenings recovered cDNA clones containing a unique amino terminal open-reading frame (A/B domain) for each gene, most similar to insect B1 EcR and USP1/RXR isoforms. Splicing variants in the UpRXR ligand-binding domain were also identified, in a region critical for folding of Drosophila and lepidopteran USP. UpEcR and UpRXR proteins were able to associate, and both are required for binding to an ecdysteroid HRE; these interactions were not hormone-dependent. Ribonuclease protection assays (RPA) were conducted using A/B domain and 'common' (C or E) domain probes on RNA isolated from various stages of regenerating limb buds and ovaries. For several of the limb bud and ovarian stages examined, the relative level of A/B domain sequence protected was significantly less than common domain suggesting alternative amino terminal isoforms other than those isolated through cloning. This is the first report of UpEcR and UpRXR transcription during ovarian maturation, implicating the ovary as a potential target for hormonal control in Crustacea.

Comparative endocrinology in the 21st century.

Hormones coordinate developmental, physiological, and behavioral processes within and between all living organisms. They orchestrate and shape organogenesis from early in development, regulate the acquisition, assimilation, and utilization of nutrients to support growth and metabolism, control gamete production and sexual behavior, mediate organismal responses to environmental change, and allow for communication of information between organisms. Genes that code for hormones; the enzymes that synthesize, metabolize, and transport hormones; and hormone receptors are important targets for natural selection, and variation in their expression and function is a major driving force for the evolution of morphology and life history. Hormones coordinate physiology and behavior of populations of organisms, and thus play key roles in determining the structure of populations, communities, and ecosystems. The field of endocrinology is concerned with the study of hormones and their actions. This field is rooted in the comparative study of hormones in diverse species, which has provided the foundation for the modern fields of evolutionary, environmental, and biomedical endocrinology. Comparative endocrinologists work at the cutting edge of the life sciences. They identify new hormones, hormone receptors and mechanisms of hormone action applicable to diverse species, including humans; study the impact of habitat destruction, pollution, and climatic change on populations of organisms; establish novel model systems for studying hormones and their functions; and develop new genetic strains and husbandry practices for efficient production of animal protein. While the model system approach has dominated biomedical research in recent years, and has provided extraordinary insight into many basic cellular and molecular processes, this approach is limited to investigating a small minority of organisms. Animals exhibit tremendous diversity in form and function, life-history strategies, and responses to the environment. A major challenge for life scientists in the 21st century is to understand how a changing environment impacts all life on earth. A full understanding of the capabilities of organisms to respond to environmental variation, and the resilience of organisms challenged by environmental changes and extremes, is necessary for understanding the impact of pollution and climatic change on the viability of populations. Comparative endocrinologists have a key role to play in these efforts.

EST library sequencing of genes expressed during early limb regeneration in the fiddler crab and transcriptional responses to ecdysteroid exposure in limb bud explants.

We have constructed directional and randomly primed cDNA libraries from mRNAs isolated during progressive stages of fiddler crab (Uca pugilator) limb regeneration. Data from these libraries are being assembled into an on-line database (http://www.genome.ou.edu/crab.html) that is both BLAST and keyword searchable; the data set is also available through GenBank. The first characterized library was made from mRNA isolated 4 days post-autotomy, when the first sign of morphological differentiation, cuticle secretion, is observed. Analysis of 1698 cDNA clones led to assignment of 473 contigs and 417 singlets, for a total of 890 sequences. Of these, ∼86% showed no assignments to characterized genes on database searching, while 14% could be assigned to a known ortholog in the COG (Clusters of Orthologous Groups) database. BLAST searches to specific protein domains in the Gene Ontology database led to assignments for ∼40% of the assembled sequences. Sequence similarity searches of other crustacean EST databases produced hits to 13-30% of the Uca query sequences. The ESTs include several genes that may be potentially ecdysteroid-responsive, such as homologs to chaperone proteins and cuticle protein genes, as well as homologs to arthropod proteins involved in retinoid/terpenoid metabolism. We have tested 3 potential candidate genes for their ability to be induced by ecdysteroid in limb bud explants; an arthropodial cuticle protein gene, and the nuclear receptor genes EcR and RXR. A subset of early blastemal limb buds (8 days post autotomy) show a positive response to ecdysteroid by 1-1.5 h, followed by a decrease in transcript abundance at longer periods of sustained incubation. Later stage buds (12 days post autotomy-late premolt) show decreases in steady-state mRNA levels by 1.5 h, or are completely refractory to ecdysteroid exposure.