Penny M. Hopkins

Cloning of crustacean ecdysteroid receptor and retinoid-X receptor gene homologs and elevation of retinoid-X receptor mRNA by retinoic acid.

We report the cloning and analysis of ecdysteroid receptor (bpEcR) and retinoid-X receptor (UpRXR) cDNA homologs from the fiddler crab Uca pugilator. The deduced amino acid sequence of this crustacean EcR most closely resembles the insect EcRs within the DNA binding and ligand binding domains (LBDs). For UpRXR, the DNA binding domain (DBD) shares greatest identity to the insect USPs. The ligand binding domain, however, is closer to vertebrate RXRs but may have a nonfunctional AF-2 domain. Probes derived from these clones were used to examine transcript levels in blastemas during early limb regeneration. Both UpEcR and UpRXR transcripts were detected in low amounts 1 day after limb loss, but increased during the next 4 days. Immersion of crabs in sea water containing all-trans retinoic acid increased the steady state concentrations of UpRXR transcript and altered the pattern of circulating ecdysteroids. These effects correlate with the disruptive effects of retinoic acid on blastemal differentiation observed in earlier studies.

Patterns of serum ecdysteroids during induced and uninduced proecdysis in the fiddler crab, Uca pugilator

Eyestalk-intact and eyestalkless fiddler crabs, Uca pugilator, have similar temporal patterns of circulating serum ecdysteroids during proecdysis. Both groups of animals showed two distinct transient peaks of radioimmunoassay (RIA)-active ecdysteroids. Peak 1 occurred 3 weeks prior to ecdysis and preceded the onset of rapid proecdysial limb bud growth. Peak 2 was a larger peak that occurred a few days prior to ecdysis. Thin-layer chromatography profiles of the two peaks showed at least seven RIA-active compounds common to both peaks. The relative abundance of these compounds differed between the two peaks. The role of the eyestalks in control of circulating ecdysteroids was limited to maintenance of intermolt conditions. During proecdysis, the control of circulating ecdysteroid levels was located outside of the eyestalks. There was no correlation between limb bud growth rates and serum ecdysteroid levels during proecdysis.

Limb Regeneration in the Fiddler Crab, Uca pugilator: Hormonal and Growth Factor Control

SYNOPSIS. This paper summarizes recent work on various aspects of hormonal control of regeneration in the crustacean, Uca pugilator. Hormonal control in this crab is effected by means of the crustactean steroid hormones, the ecdysteroids. New evidence is presented supporting a role for the retinoid hormones, all-trans retinoic acid and 9-cis retinoic acid, in the control of regeneration in these animals. The possible role of fibroblast growth factors in organization of the limb blastema is explored and the similarities between vertebrate and invertebrate control of regeneration are discussed.

Expression of the genes encoding the ecdysteroid and retinoid receptors in regenerating limb tissues from the fiddler crab, Uca pugilator

Using sequence information derived from the Drosophila melanogaster (Dm) ecdysteroid receptor (EcR)- and retinoid X receptor (RXR)-encoding gene homologs, we have isolated cDNA clones corresponding to the DNA-binding domains (DBD) for these two nuclear receptors from the fiddler crab, Uca pugilator (Up). Both genes appear to be represented in 1-2 copies in the Up genome, and unlike Dm, contain an intron within the DBD-encoding region. Sequence comparisons to the Dm EcR and RXR homologs indicate 76 and 82% nucleotide identity, respectively, corresponding to 6 and 4 single-amino acid substitutions which primarily cluster in the region of the molecule involved in dimerization. RT-PCR analysis indicates that both the EcR and RXR homologs are expressed during the initial stages of limb regeneration, temporally concomitant with early blastema formation and the secretion of a flexible sac cuticle at the site of limb loss.

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.

Ecdysteroid titers and Y-organ activity during late anecdysis and proecdysis in the fiddler crab, Uca pugilator.

The titer of ecdysone in hemolymph and the ratio of ecdysone to other radioimmunoassay(RIA)-active hemolymph ecdysteroids were compared to in vitro secretion of ecdysone in Y-organs removed from eyestalkless fiddler crabs at various times following eyestalk ablation. Using high-pressure liquid chromatography (HPLC) and RIA it was established that ecdysone, 20-hydroxyecdysone, and RIA-active metabolites are present in the hemolymph at the end of anecdysis and throughout proecdysis. There was little correlation between in vitro secretory activity and total ecdysone in circulation. Ratios of ecdysone and 20-hydroxyecdysone changed during proecdysis as did the percentage of total RIA activity attributable to both. Positive correlations were observed between in vitro Y-organ secretion rates and amounts of ecdysteroids extracted from hemolymph and added to incubation media.

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.