Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated ‘regulatory hotspots’ around genes closely associated with progenitor programs. To examine their functional significance, we deleted ‘hotspot’ enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.

This study presents data from the cross-reactivity analysis of purified ciguatoxin (CTX), okadaic acid (OA), and the East sphere or Fragment B-C of OA with their homologous antibodies, monoclonal antibodies to ciguatoxin (MAb-CTX) and okadaic acid (MAb-OA). The test system used was the stick enzyme immunoassay. MAb-CTX gave peak titers of 1.5ng, 10ng and 50ng respectively for CTX, East sphere and OA. Competitive inhibition analysis showed that 4ng purified CTX blocked completely MAb-CTX reaction with crude CTX, OA and East sphere of OA blocked at similar concentrations (approximately 50ng). The activity with MAb-OA in the homologous system with OA and East sphere was insignificant. This may be attributable to the improper concentrations used. The cross-reactivity between MAb-CTX with OA and its Fragment B-C may cause difficulty in the test system in its application to assess toxic fish due to ciguatoxin.

The stick enzyme immunoassay (S-EIA) using monoclonal antibody to ciguatoxin (MAb-CTX) was used to examine clinically implicated fish and to pre-screen two species of fish, Caranx sp. (ulua or jack) and Seriola dumerili (kahala or amberjack), supplied by sports fishermen. All of the clinically implicated fish from the Department of Health gave S-EIA values greater than or equal to 1.3. The Caranx sp. and Seriola dumerili considered safe (less than or equal to 1.2 value) and consumed after testing gave no false-negative results. The S-EIA procedure using MAb-CTX proved to be specific, sensitive, and simple to use in the laboratory. It also proved to be useful in screening two large carnivorous fish for ciguatoxin and related polyethers prior to consumption.

A survey for the assessment of the ciguatera problem has been determined in Puako, South Kohala, on the Island of Hawaii. This is in the area of persistent ciguateric outbreaks during the months of January through March, caused by a specific species of fish (Cheilinus rhodochrous, red rose wrasse, or po'ou). Analyses of algae, Gambierdiscus toxicus, and various species of fish, including herbivores and carnivores, gave positive indications of Puako as a potential ciguateric area. Algae associated with Gambierdiscus toxicus blooms and the dinoflagellate itself were found in transects A and D. Transects A and D showed 291 G. toxicus per gram of Tolycarpidia glomurata and 9 G. toxicus per gram of Turbinaria sp. with epiphytic Jania sp., respectively. No G. toxicus was found in transects B and C. This may be attributed to the low salinity from intrusion of freshwater in this vicinity. Examinations of the fish, kole, manini, Hawaiian kole, roi, and po'ou by the solid-phase immunoassay showed 89% of fish in the borderline and positive categories from all transects. Extracts of viscera and flesh showed high levels of toxicity in mouse (13 of 23 deaths), particularly in the viscera (gut) of both herbivores and carnivores. The guinea pig atrial analysis generally showed a few ciguatoxin-like, but most were nonciguateric type responses. The data presented in this Puako survey showed evidence of toxic fish associated with ciguatoxin-like and most probably other toxins, either polyethers or non-polyethers as yet unidentified.

Following the death of two Atlantic dolphins in a lagoon in March of 1989, the Hawaiian fishes in the lagoon were examined as a potential source of toxin(s). This study reports the findings of the causitive toxin(s) involved, utilizing the stick enzyme immunoassay (S-EIA) and the mouse and guinea pig atrium assays. The S-EIA proved effective in screening the toxic fishes (mullet, wrasse, manini, and aholehole). Following extraction, the major toxin was found in the viscera of these fishes, as confirmed in the mouse assay. The most toxic level was shown in the viscera of the mullet (13.2 mouse units/mg of extract). The viscera of the wrasse, aholehole, and manini also showed high levels of the toxic substance. The guinea pig atrium assay showed the presence of a potent Na+ channel inhibitor, characteristic of tetrodotoxin and saxitoxin. The toxin was also demonstrated in low levels in the dolphin liver and gut content and in the sand and algae extracts from the lagoon. This is the first report of this type of toxin in Hawaii.