Nitrogen can substitute helium as a mobile phase in the analysis of wastewater treatment matrices for persistent organic pollutants by gas chromatography - electron capture detection system.

Municipal wastewater treatment plants are required to monitor persistent organic pollutants (POPs) in their wastewater treatment related discharges and to assess the impact of the discharges on the environment and public health. One tool for monitoring chlorinated organic pollutants particularly is a gas chromatographic (GC) system coupled to a pair of halogen-specific electron capture detectors (ECDs) with helium (He) as the mobile phase. He supplies, however, has become inconsistent and unreliable lately. In its place, N2 gas is evaluated in this study as a potential substitute for He in quantifying organochlorine pesticides, polychlorinated biphenyls, chlordane congeners and toxaphene in wastewater treatment related matrices (influent, effluent, benthic sediment, mussel tissue, and biosolids/sludge). N2 is inert, inexpensive and requires no additional hardware to incorporate into the basic functions of a GC-ECD. Our results show that, with the usual data quality controls (blank, laboratory control, matrix spike/duplicate and proficiency testing samples, and the fact that certified reference materials data met requirements), N2 can replace He for regulatory purposes. And when necessary, the N2-based retention times (tN) can be predicted reliably from He-based retention times (tHe), irrespective of column chemistry or POPs (here: tN = 1.90tHe + 0.04, R2 = 0.996).

Application of supervised learning classification modeling for predicting benthic sediment toxicity in the southern California bight: A test of concept.

Benthic sediment toxicity is linked to harmful effects in marine organisms and humans, and an understanding of the link would require, in part, a comprehensive and exhaustive analysis of sediment toxicity data already in hand. One tool which could aid in the process is machine learning (ML), a supervised classification modeling technique that has transformed how actionable insight are acquired from large datasets. The current study is a test of concept in which an ML classifier is sought that can accurately extrapolate the characteristics of a 5437 California-wide coastal training dataset (assembled from 1635 samples) to predict sediment toxicity in southern California bight (SCB). Twelve classifiers were trained to recognize sediment toxicity using 70 % of the dataset and among them, a Gradient Boosting Classifier (GBC) model using latitude, longitude, and water depth was found to be the most accurate at predicting toxicity (83 %). Among the variables, latitude was found to be the most significant driver of prediction by GBC in this test ecosystem. The performance of the model was verified with the remaining 30 % of the dataset and found to be 83 % accurate. Presented with 884 unfamiliar data points assembled from 854 measurements at 346 stations across SCB, GBC was 87 % accurate post-training, thus demonstrating a role supervised learning can play in the southern California environmental analytics.

Harmful algae blooms: an analysis of recent spatiotemporal trends on California's inland waterbodies.

Harmful algae blooms (HABs) are unwelcome annual events on waterbodies in California (USA). The unseemly sights of lifeless fish drifting ashore and the mammalian illnesses resulting from close contacts with HABs once in ahile are telltale signs of their presence. California as such tracks HABs closely for immediate public warnings. That said, a lack of statewide monitoring standards for producing comparable and high-quality data, however, hinders a deeper insight into HABs on California's inland waterbodies. In their absence, the current study demonstrates the utility of empirical dataset in advancing an understanding of spatiotemporal distribution of HABs on inland waterbodies. With a 2016-2021 mostly observational data, the questions asked are whether HABs incidences are expanding in California with time, whether there are localities more prone to HABs than others, and whether bivariate relationships exist between HABs reports and population density, or waterbody size, land area and geographical location. Overall, the frequency with which HABs are observed is found to increase statewide, an increase not correlated with population density, but with land area and inland waterbody size. Results also show that North Coast, Central Valley, and San Diego are the most affected regions of California. Also revealed by this mostly observational data were recent HABs spikes in the January months (California winter) which, if proven, may be signaling an emerging year-round problem that California will have to contend with.

Multivariate analysis of heavy metals content of beef from Soroti, Uganda.

Information about food hygiene and quality in the sub-Saharan African countries remains scarce at a time when many of their citizenry are beginning to acquire the much coveted middle income status. Confounding this are challenges linked to monitoring on a continuous basis the safety of food produced by such lucrative industries as the beef industry. The objective of the current study was to initiate a process of encouraging changes in the status quo, by showing how a first step in that direction might look like. Using heavy metal contents of representative beef samples from butcheries in Soroti, Uganda, typical of a sub-Saharan country, we demonstrate how relationships and common sources of metals in food could be identified in a multivariate space. Beef samples from 40 sites were analyzed by atomic absorption spectrometry for iron (Fe), zinc (Zn), nickel (Ni), chromium (Cr), lead (Pb), copper (Cu), cobalt (Co) and cadmium (Cd). The study showed that all beef samples contained these metals, the extent of which were in the order: Fe > Zn >> Ni, Cr > Pb > Cu, Co > Cd. By correlation analysis, the pairs Ni and Cr, Cd and Co, Ni and Fe or Cr and Fe were found to be most likely coming from similar sources. At least three distinct characteristics of beef consumed in Soroti were also found, a distinction perhaps arising from three major categories of feedlots used to raise donor cattle. The incremental risk of children or adults developing cancer over a lifetime was estimated and found to fall into three categories, two of which are separately explained by the presence of Cr or Ni. The sources of these metals remain a matter of speculation on our part. More studies are needed to determine these sources and to understand the nature of cancer risk in the three categories of beef identified here.

A Morphometric Assessment of the Condition of White Croakers (Genyonemus Lineatus) and Queenfish (Seriphus Politus) with Access to a Wastewater Outfall and a DDT/Polychlorinated Biphenyl Superfund Site: A Los Angeles Outer Harbor Case Study.

Sediments and water columns in the Los Angeles Outer Harbor (CA, USA), a major port behind a breakwater, contain DDTs and polychlorinated biphenyls (PCBs) from a nearby Superfund site, and contaminants brought in by ships, boats, stormwater, a river, and a wastewater outfall. White croaker and queenfish, a bottom feeder and a water column feeder, respectively, are two bioindicators for this marine ecosystem. Their condition as populations of fish is assumed to be robust for this role at all times. The present study tests this benign assumption amid progressively increasing DDT/PCB levels in their tissues. The results, as shown by progressively shrinking gonads, show a less than robust white croaker population particularly. Although the males are generally larger than the females, the length (standard [SL] or total [TL]) and body mass (BM) of 80 white croakers collected over 8 years were found to be similar irrespective of gender (177 mm, 212 mm, and 114 g, respectively). Queenfish (67) did not show such similarity over the same period (female: 152 mm SL, 177 mm TL, 56 g BM; male: 145 mm SL, 172 mm TL, 50 g BM). The site-specific expressions/values capturing the current conditions of these fish populations are SL = 0.835[TL] - 1.68 (r2 = 0.914, n = 68) and SL = 0.891[TL] - 8.88 (r2 = 0.961, n = 50) for white croaker and queenfish, respectively. In the allometric growth equation BM = a[SL]b , a and b are 2.83 and 2.49 × 10-4 (r2 = 0.817) for white croakers, and 6.10 and 2.73 × 10-5 (r2 = 0.825) for queenfish, respectively. The relative coefficients of condition Kn are 0.97 ± 0.07 and 1.01 ± 0.12 for white croakers and queenfish, respectively. Molecular-level studies are needed to establish definitively the links between DDT/PCB bioaccumulation in fish tissues and the robustness of the fish populations. Environ Toxicol Chem 2022;41:410-425. © 2021 SETAC.

Comparison of metal bioaccumulation in crop types and consumable parts between two growth periods.

A high proportion of populations in most developing countries live below the poverty line and those near refuse grounds resort to dumpsite farming to grow food. Consequently, high levels of waste-derived contaminants are found in crops consumed by these people. This study investigates the extent to which crops cultivated on the Mbale dumpsite (Uganda) were contaminated by 11 metals and 2 non-metals: iron (Fe), aluminum (Al), zinc (Zn), manganese (Mn), copper (Cu), mercury (Hg), lead (Pb), nickel (Ni), cobalt (Co), cadmium (Cd), selenium (Se), chromium (Cr), and arsenic (As). We investigated how element bioaccumulation in crops was influenced by the growth period (short- and long-term crop maturity). The short-term crops were Zea mays and Amaranthus cruentus, whereas the long-term crops were Manihot esculenta, Colocasia esculenta, Musa acuminata, Carica papaya, Coffea arabica, and Saccharum officinarum. Results showed that nine metals were present at concentrations above World Health Organization/Food and Agriculture Organization (WHO/FAO) food safety recommendations and hence may pose health risks to consumers. In this study, leaves contained higher metal concentrations than other analyzed consumable parts. Pb and Co were found at higher concentrations in leaves of short-term crops than in long-term crops. Among short-term crops, only Z. mays seeds contained permissible metal concentrations by WHO/FAO standards. The growth period was also found to influence metal bioaccumulation in crop types. Pb, Co, Fe, Al, and Cu concentrations were significantly higher in the short-term crops than in long-term crops, while Mn, Ni, and Cr concentrations were higher in long-term crops than in short-term crops. Overall, public awareness about the health risks associated with consuming short-term leafy crops grown on dumpsites should be improved to reduce toxic metal exposure. While implementing such a campaign, the food supply of individuals whose survival depends on such crops should not be jeopardized. Therefore, farmers need alternative farming areas outside dumpsites. Integr Environ Assess Manag 2022;18:1056-1071. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Indirect assessment of biomass accumulation in a wastewater-based Chlorella vulgaris photobioreactor by pH variation.

Algae bloom in coastal waters is partly supported by residual nutrients in treated wastewater (WW) released from coastally located treatment plants. In response, a Chlorella vulgaris-based photobioreactor was recently proposed for lowering nutrient levels in WW prior to release. However, the solution requires maintaining biomass accumulation to within a photobioreactor capacity for optimum operation. For high density Chlorella vulgaris suspensions, this is easily done by monitoring turbidity increase, a property directly related to biomass accumulation. For low density suspensions however, direct turbidity measurement would require a cumbersome process of concentrating large volumes of Chlorella vulgaris suspensions. Here, we demonstrate that by measuring pH of the suspensions, turbidity (T) can be estimated indirectly by the following wastewater-dependent expression: pH = aT + pH0, hence avoiding the need to concentrate large volumes. The term pH0 is the initial pH of the suspensions and a, a wastewater-dependent constant, can be computed independently from a = - 0.0061*pH0 + 0.052. In the event %WW is unknown, the following wastewater-independent Gaussian expression can be used to estimate T: pH = 8.71*exp(- [(T - 250)2]/[2*1.26E05]). These three equations should offer an avenue for monitoring the turbidity of dilute Chlorella vulgaris suspensions in large, stagnant municipal Chlorella vulgaris-based wastewater treatment system via pH measurements.

Multivariate Analysis of Sediment Toxicity in an Ocean Ecosystem: A Southern California Bight Case Study.

Contaminated sediments can negatively affect aquatic organisms and beneficial uses of coastal regions. Monitoring programs typically collect many indicators of sediment toxicity, yet multivariate approaches that comprehensively evaluate data across heterogeneous spatial environments are frequently not performed. In this paper, we explore a multivariate approach to show that a list of suspected drivers of sediment toxicity to native Mytilus galloprovincialis (mussel) and Eohaustorius estuarius (a marine amphipod) population can be narrowed down without excluding samples, and that redundancies in sampling sites can be identified and isolated. Using a 153 × 28 data matrix assembled from a southern California-wide bight monitoring program, we demonstrate by this approach that Port of Los Angeles (PLA) and San Diego Bay (SDB) contained the most toxic sediments in the bight in 2008, the nature of which was unique to each locality. (Note: Little toxicity was observed here in 2013 and 2018.) In PLA sediments, mussels were more affected than amphipods, with higher survivability associated with low Hg and Sn levels. Conversely, amphipods had higher mortality than mussel embryos in SDB sediments, with higher survivability associated with low Be and Co levels. Nitrogen, organic content, and finer sediment particles were not related to the survivability of these organisms.

Descriptive Analysis of Heavy Metals Content of Beef From Eastern Uganda and Their Safety for Public Consumption.

In this study, we initiated an effort to generate information about beef safety in Uganda. Our entry point was to assess by atomic absorption spectrophotometry the levels of essential elements copper (Cu), cobalt (Co), iron (Fe) and zinc (Zn), and non-essential elements lead (Pb), chromium (Cr), nickel (Ni), and cadmium (Cd) in 40 beef samples collected from within and around Soroti (Uganda). The information was used to evaluate the safety of consuming such beef against the World Health Organization (WHO) limits. The latter was accomplished by (i) estimating the daily intake (EDI) of each metal in the study area, (ii) modeling the non-cancer health risk using the target hazard quotient (THQ) and (iii) modeling the cancer risk using the incremental lifetime cancer risk (ILCR). The study finds that the mean concentrations (±95% CI) and EDI were in the order of Fe > Zn > Cr > Ni > Pb > Co > Cu > Cd. Cancer risk was found to be due to Ni > Cr > Cd > Pb and significantly higher in children than adults. The latter particularly demonstrates the importance of Ni poisoning in the study area. Overall, while essential elements in our beef samples were below WHO limits (hence no health risks), non-essential elements had high health and cancer risks due to higher levels of Cr and Ni.

Quantifying semi-volatile organic contaminants in solution by internal standard addition method requires prompt addition of the internal standards.

The precision and accuracy of quantifying semi-volatile organic compounds (SVOCs) in solution by GC/MS, particularly when volume errors are unpredictable or difficult to control, are improved by utilizing internal standards (IS). Not obvious though is the extent to which timing IS addition affects measurement. To illustrate this fact, the mean concentrations of 60 SVOCs (40 or 80 µg/mL) in two identical solutions into which IS were added at different times are compared in this study. In one solution, IS were added promptly on preparation (reference); in the other, IS were added after 36 days of incubation (treatment). To investigate the role that temperature might play here as well, equal fractions of each solution were incubated at - 20 °C, 4 °C or 22 °C. Results, as determined by one-way ANOVA, show that there were no differences between the reference solutions at the beginning and after 36 days (F3,236 = 0.244, p = 0.865), but that significant differences exist between the reference solutions collectively and the treatment irrespective of temperature (F6,413 = 6.76, p = 1.99e-06). These results, confirmed by a post hoc analysis, suggest that uncertainty is introduced into SVOC quantitation when internal standards are not added promptly into SVOCs solutions on preparation.

Disappearance kinetics of 56 toxic semi-volatile organic compounds under common storage conditions.

Synthetic semi-volatile organic compounds (SVOCs) are widely distributed in the environment due to their ever expanding uses. But because some are hazardous, their presence in matrices such as indoor air, wastewater, fish tissue and marine sediment samples (where they could adversely impact public health and the environment) is carefully monitored. To do so, samples are collected, stabilized for transport to laboratories (which are often off-site) and purified for analysis. In-between purification and analysis, the stability of SVOCs in storage is largely assumed. In this study, we test this assumption by assessing the collective stability of 56 SVOCs (amines, halo ethers, nitrobenzenes, phenols, phthalate esters, polycyclic aromatic hydrocarbons and chlorinated compounds) in mixtures stored at -20 °C, 4 °C or 22 °C over a 53 day period. Results show that (i) SVOCs are stable in darkened Amber vials, (ii) that constant temperature is more important than fluctuating temperature in maintaining the integrity of samples in storage, and (iii) that the collective disappearance of SVOCs in colorless 1 mL vials follow first-order kinetics with half-lives (in days) of 28 ± 8, 26 ± 3 and 31 ± 7 at -20 °C, 4 °C or 22 °C, respectively. PAHs (and phthalate esters) were mostly stable with dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene and 1,12-benzoperylene showing the longest half-lives (57 ± 2 days at -20 °C and 65 ± 6 days at 4 °C, respectively). The degree of substitution on phenols by deactivating groups appears to introduce stability differences. While mono-substituted phenols (chloro and nitro) were sensitive to temperature changes, di- and tri-substituted phenols (dinitro, dichloro and trichloro) were not. Overall, light was found to be the most important factor in the disappearance of SVOCs in colorless vials.

Examining the correlation between quantifiable SVOCs and organic carbon content or particulate size in benthic sediments as a function of ocean stratum.

The raw analytical concentration values of semi-volatile organic compounds (SVOCs) in ocean sediments do not necessarily reflect the bioavailable fractions of SVOCs in the sediments due to factors such as the total organic carbon content of sediments (TOC) and the percentage of fine particles in sediments (% fines) as they are believed to affect the extraction efficiency of SVOCs from sediments. Corrective actions are therefore taken to minimize their influence on measurements. In doing so, a broad and uniform correlation is assumed to exist between the 'native' levels of SVOCs and TOC or % fines across ocean strata. However, the validity of this blanket assumption is not yet verified. In this study, we examined the strength of the assumption using DDTs, PAHs and PCBs levels in sediments from Santa Monica Bay (SMB), California, USA. (The distribution patterns of these SVOCs in SMB are known and reproducible for quality assurance.) As our results show, a uniform correlation between SVOC levels and TOC or % fines across strata is mostly absent. For example, PAH and PCB levels show negative correlation with TOC or % fines, and only in canyon sediments, DDT levels correlate positively with both TOC and % fines across at least three strata. Furthermore, the distribution of PAH molecules appears to be controlled by molecular size with smaller PAHs being found almost exclusively in the canyons. Our finding here, being the first of its kind, suggests that more work is needed to clarify the reporting of SVOC levels in ocean sediments.

To freeze, or not to freeze: the impact of subzero temperature on quantifying organic contaminants in ocean sediments.

Assessing the extent of ocean bed contamination by anthropogenic organic chemicals requires collecting and preserving the native state of sediments. The latter is particularly important since most sediment analyses are performed away from the sites of samples collection. Preservation, however, is presumptuous since commonly used sediment handling practices such as freezing are structurally disruptive, the impacts of which are not well understood. In this study, the impact of freezing on quantifying the total organic carbon (TOC) contents and the extent of PAH, DDT and PCB contamination in 17 split-paired sediment samples was investigated. The samples were collected from the Santa Monica Bay, California (USA). One-half of each split-pair was frozen at -20 °C and the other half was refrigerated at 4 °C for up to 11 days. The results suggest that no significant differences exist between the frozen and the refrigerated datasets for PCBs (F1,28 = 4.01, p > 0.05), DDTs (n = 16, t-Stat < t-Critical, p > 0.05) or TOC (n = 16, t-Stat < t-Critical, p > 0.05). The results however show that less PAHs were detected in the frozen sediments (F1,24 = 8.18, p < 0.05) than in the refrigerated sediments; the larger PAHs were affected the most. Interestingly, while benzo[a]pyrene, a large PAH molecule, was affected by this apparent temperature-induced difference, its structural isomer, benzo[e]pyrene, was not. Even more interesting was the finding that while non-coplanar PCBs were affected similarly, the coplanar PCBs were not. Overall, sediment freezing within this study's timeframe appears to offer little contextual advantage over sediment refrigeration.

Applying multivariate analysis to characterize waragi spirits from Acoli, Uganda, by their metal contents.

Quality control during spirits production and means of authenticating or verifying sources of spirits in the sub-Saharan region of Africa are limited due to lack of resources and the scientific acumen required to develop methodologies for characterizing spirits. However, the increasing needs to protect consumers from negligence, or willful contamination of spirits in this region underscores the urgency with which growth in this area must happen. In this paper, we describe a multivariate statistical framework upon which characterization, identification and authentication of spirits could be developed. The framework exploits the unique chemical fingerprints of spirits with the goal of accomplishing three functions simultaneously: the detection of class differences, the authentication of spirits and the verification of sources. In a test case using the metal contents of 17 Ugandan spirits, this framework shows (i) that a class of unrecorded spirits known locally as Lira-Lira can be singled out from other spirits by their Cu contents, (ii) that localities from where the Lira-Lira spirits were purchased can be resolved to within 8 km by cluster analysis and principal component analysis, (iii) that cluster analysis loadings and scores, placed side-by-side, can pair spirits and their unique discriminating contaminants directly, (iv) that the most important metals for authenticating 13 spirits, source verification and production methods are Al, Sr, Ba, Mn, Zn and Cu (high concentration variability across samples is the qualifying factor), (v) that common sources of contamination can be detected by Pearson correlation analysis (this study finds that Sn/Cd, Pb/Cr, Tl/Cr, Pb/Ni or Cu/Ag as well as the triad Se/As/Ni in the 13 Ugandan spirits are from similar sources), and (vi) that inconsistency in spirits production can be detected with empirical data. Such rudimentary solutions to characterizing spirits have never been offered to the sub-Saharan countries.

Assessing the health risks of consuming 'sachet' alcohol in Acoli, Uganda.

The increased mortality rate among the Acoli people of northern Uganda is anecdotally blamed on excessive consumption of cheap and widely available sachet-packaged alcohol in the region. In this paper, we quantify this perceived association by determining statistically the health risks associated with ingesting 20 heavy metals in 17 popular spirits products consumed in Acoli. Thirteen of these products were industrially packaged in sachets (locally known as 'sachet,' waragi, arege or moo lyec) and four were locally produced Lira-Lira spirits from Bolo, Awere and Teso Bar in the region and Nsambya in southern Uganda. A Scottish whisky purchased in San Diego (USA) was our reference. Risk assessment was performed according to standardized protocols developed by the United States Environmental Protection Agency (US EPA). Our results show that a strong correlation indeed exists between health risks and ingestion of spirits in Acoli. At >2.5 sachets/day for 240 day/year over a lifetime for example, the risk of developing cancer due to exposure to As, Pb and Cr alone is 1 in 102,041. This estimate excludes ethanol, a known carcinogen, and 17 heavy metals also studied due to lack of their cancer slope factors. The primary non-cancer related health risk factor in all samples tested is ethanol with unacceptably high health index of four. The Lira-Lira spirits, with 100-6000% copper above the US EPA limit for intake by oral ingestion in water, would be the 'cleanest' without copper and at par with the Scottish whisky. Collectively, we find that no amount of alcohol consumed in Acoli is safe. Preventive measures are therefore recommended to reduce mortality in Acoli in particular, and in Uganda in general. These measures should include public education, better public policies, creating productive economic activities other than brewing alcohol, and social activities that engage people away from drinking.

The effect of a massive wastewater discharge on nearshore ocean chemistry.

An opportunity to study the effect of a massive wastewater discharge on a nearshore ocean environment arose in 2015 over a 42-day period when the City of Los Angeles diverted 9.4 billion gal of treated wastewater effluent from an outfall located 5 mi in the Pacific Ocean to a 1-mi backup in the Santa Monica Bay (SMB). SMB is a heavily used waterbody and is home to many marine organisms. To understand the impact of this diversion on human health and on SMB ecosystem, samples of the wastewater effluent, the receiving seawater, and sediments from around the backup outfall were analyzed, among others, for metals, semivolatile organic compounds (SVOC), nutrients, and total organic carbon (TOC) during the diversion project. Results show that these parameters were present at levels below local water quality guidelines (i.e., not enough to cause health or environmental concerns). In the effluent, metal levels were < 10 µg/L except Zn (23 ± 9.9 µg/L); phosphate, ammonia-nitrogen, organic-nitrogen, and TOC levels were 3.48 ± 0.37, 42.7 ± 5.3, 4.7 ± 1.4, and 19.0 ± 4.9 mg/L, respectively. In seawater and sediments around the backup outfall, metal levels were < 1.5 µg/L and < 25 mg/kg, respectively. Apart from 4,4'-DDE, SVOCs were not detected in sediments. To assess whether changes to native levels of pollutants in SMB occurred due to the diversion project, pre-diversion and post-diversion datasets were compared statistically. No significant differences were found between the two datasets (p > .05, paired t test), meaning the diversion did not change the SMB chemistry.

An empirical model of Onecut binding activity at the sea urchin SM50 C-element gene regulatory region.

Studying the formation of endoskeleton in many species is complex and difficult. The sea urchin embryo offers an unparalleled platform for understanding this process because of the ease with which its skeletogenic mesenchyme cells can be manipulated. In this study, preliminary evidence from biochemical studies towards understanding the role of the Onecut transcription factor during sea urchin skeletogenic mesenchyme cell specification is presented. Based on the evidence, an empirical model is proposed showing how Onecut, together with associated co-factors, may be using the C-element of the SM50 gene regulatory region in advance of the sea urchin Strongylocentrotus purpuratus spicule development. In the model, Onecut recognizes and binds the DNA sequence CATCGATCTC in the C-element without temporal restriction. Onecut then utilizes different sets of co-factors to switch from its unknown function early in development (four cell stage to the mesenchyme blastula stage), to its known role in the oral-aboral boundary thereafter. At the writing of this report, definitive evidence as to whether the "early" factors are expressed in all cells except the micromere lineages, or whether the "late" factors are expressed in micromere descendants or ectodermal precursors only are lacking. The former would suggest a possible Onecut repression function for the early co-factors outside the micromere lineages; the latter scenario would suggest a Onecut activation function for the late co-factors in the presumptive ciliary band.

Expression of AmHNF6, a sea star orthologue of a transcription factor with multiple distinct roles in sea urchin development.

The sea urchin transcription factor SpHNF6 is an early activator of differentiation genes in skeletogenic lineages and regulatory genes in the oral ectoderm. We report here the cloning and the expression of an orthologue of this gene, AmHNF6, from the sea star Asterina miniata. The vertebrate and the echinoderm hnf6 and onecut genes belong to the novel ONECUT homeo domain class of transcription factors. In blastula stage sea star embryos, AmHNF6 is expressed everywhere except around the vegetal pole. As is observed in sea urchin, by the end of gastrulation, the expression of AmHNF6 is distinctly localized to the ciliary bands. This terminal phase of expression has remained unchanged since the divergence of these two taxa half a billion years ago.

SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis.

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression.

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.