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In the nematode, C elegans, the divergent GATA-type transcription factors MED-1 and MED-2 are encoded by an unlinked, redundant pair of intronless genes. The med-1,2 genes are among the first to be activated in the embryo and are critical for the specification of the 7-cell stage MS (mesoderm) and E (endoderm) precursor cells. We have previously shown that the binding site recognized by MED-1 is a noncanonical RAGTATAC site that is not expected from the resemblance of its single C4-type zinc finger to those of other known GATA factors, which recognize the consensus HGATAR. To date, no MED-like zinc fingers have been described outside of C. elegans. In order to understand the evolution of these transcription factors, and the evolution of gene networks that specify early cell fates in Caenorhabditis, we have identified med sequence homologs in the related nematodes C. briggsae and C. remanei. While C. briggsae encodes two med-like genes similar to C. elegans, we find evidence for seven distinct med-like genes in C. remanei. Somewhat unexpectedly, the coding regions of all med genes appear to lack introns. We report that the med homologs have similar expression in their respective species. We further show that the C. briggsae homologs, and at least five of the seven C. remanei homologs, can fully complement the embryonic lethal phenotype of a C. elegans med-1,2(-) strain. We conclude that Med function and expression have been conserved over tens of millions of years of evolution, and that there may be a mechanism that selects against the acquisition of introns in these genes. (c) 2005 Elsevier Inc. All rights reserved.

Author(s): R. Lu [1, 4]; M. Maduro [2, 4]; F. Li [1, 3]; H. W. Li [1]; G. Broitman-Maduro [2]; W. X. Li [1]; S. W. Ding (corresponding author) [1, [...]

Objective: Assessing excess deaths from benchmarks across causes of death during the first wave of the COVID-19 pandemic and identifying morbidities most frequently mentioned alongside COVID-19 deaths in the death record., Methods: Descriptive study of death records between 11 March 2020 and 27 July 2020, from the New York City Bureau of Vital Statistics. Mortality counts and percentages were compared with the average for the same calendar period of the previous 2 years. Distributions of morbidities from among forty categories of conditions were generated citywide and by sex, race/ethnicity and four age groups. Causes of death were assumed to follow Poisson processes for Z-score construction., Results: Within the study period, 46 563 all-cause deaths were reported; 132.9% higher than the average for the same period of the previous 2 years (19 989). Of those 46 563 records, 19 789 (42.5%) report COVID-19 as underlying cause of death. COVID-19 was the most prevalent cause across all demographics, with respiratory conditions (prominently pneumonia), hypertension and diabetes frequently mentioned morbidities. Black non-Hispanics had greater proportions of mentions of pneumonia, hypertension, and diabetes. Hispanics had the largest proportion of COVID-19 deaths (52.9%). Non-COVID-19 excess deaths relative to the previous 2-year averages were widely reported., Conclusion: Mortality directly due to COVID-19 was accompanied by significant increases across most other causes from their reference averages, potentially suggesting a sizable COVID-19 death undercount. Indirect effects due to COVID-19 may partially account for some increases, but findings are hardly dispositive. Unavailability of vaccines for the time period precludes any impact over excess deaths. Respiratory and cardiometabolic-related conditions were most frequently reported among COVID-19 deaths across demographic characteristics., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cells in a developing animal embryo become specified by the activation of cell-type-specific gene regulatory networks. The network that specifies the gut in the nematode Caenorhabditis elegans has been the subject of study for more than two decades. In this network, the maternal factors SKN-1/Nrf and POP-1/TCF activate a zygotic GATA factor cascade consisting of the regulators MED-1,2 → END-1,3 → ELT-2,7, leading to the specification of the gut in early embryos. Paradoxically, the MED, END, and ELT-7 regulators are present only in species closely related to C. elegans , raising the question of how the gut can be specified without them. Recent work found that ELT-3, a GATA factor without an endodermal role in C. elegans , acts in a simpler ELT-3 → ELT-2 network to specify gut in more distant species. The simpler ELT-3 → ELT-2 network may thus represent an ancestral pathway. In this review, we describe the elucidation of the gut specification network in C. elegans and related species and propose a model by which the more complex network might have formed. Because the evolution of this network occurred without a change in phenotype, it is an example of the phenomenon of Developmental System Drift.

The C. elegans elt-3 gene encodes a GATA transcription factor that is expressed in the hypodermis and has roles in hypodermal specification and regulation of collagen and stress response genes. The gene encodes short and long isoforms, ELT-3A and ELT-3B respectively, that differ upstream of their DNA-binding domains. Previous work showed that ELT-3A can specify hypodermal cell fates when forcibly overexpressed throughout early embryos. We recently showed that the ELT-3B orthologue from the distantly related species C. angaria can specify endodermal fates when forcibly overexpressed in C. elegans. Here, we show that C. elegans ELT-3B can also specify endoderm., (Copyright: © 2023 by the authors.)

Endoderm specification in Caenorhabditis elegans occurs through a network in which maternally provided SKN-1/Nrf, with additional input from POP-1/TCF, activates the GATA factor cascade MED-1,2→END-1,3→ELT-2,7. Orthologues of the MED, END and ELT-7 factors are found only among nematodes closely related to C. elegans, raising the question of how gut is specified in their absence in more distant species in the genus. We find that the C. angaria, C. portoensis and C. monodelphis orthologues of the GATA factor gene elt-3 are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, Can-pop-1(RNAi), Can-elt-3(RNAi) and a Can-elt-3 null mutation result in a penetrant 'gutless' phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. Forced early E lineage expression of Can-elt-3 in C. elegans can direct the expression of a Can-elt-2 transgene and rescue an elt-7 end-1 end-3; elt-2 quadruple mutant strain to viability. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simpler POP-1→ELT-3→ELT-2 gene network., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Objectives. To assess if historical redlining, the US government's 1930s racially discriminatory grading of neighborhoods' mortgage credit-worthiness, implemented via the federally sponsored Home Owners' Loan Corporation (HOLC) color-coded maps, is associated with contemporary risk of preterm birth (< 37 weeks gestation). Methods. We analyzed 2013-2017 birth certificate data for all singleton births in New York City (n = 528 096) linked by maternal residence at time of birth to (1) HOLC grade and (2) current census tract social characteristics. Results. The proportion of preterm births ranged from 5.0% in grade A ("best"-green) to 7.3% in grade D ("hazardous"-red). The odds ratio for HOLC grade D versus A equaled 1.6 and remained significant (1.2; P  < .05) in multilevel models adjusted for maternal sociodemographic characteristics and current census tract poverty, but was 1.07 (95% confidence interval = 0.92, 1.20) after adjustment for current census tract racialized economic segregation. Conclusions. Historical redlining may be a structural determinant of present-day risk of preterm birth. Public Health Implications. Policies for fair housing, economic development, and health equity should consider historical redlining's impacts on present-day residential segregation and health outcomes.

Background: "Pneumonia and influenza" are the third leading cause of death in New York City. Since 2012, pneumonia and influenza have been the only infectious diseases listed among the 10 leading causes of death in NYC. Most pneumonia and influenza deaths in NYC list pneumonia as the underlying cause of death, not influenza. We therefore analyzed death certificate data for pneumonia in NYC during 1999-2015., Methods: We calculated annualized pneumonia death rates (overall and by sociodemographic subgroup) and examined the etiologic agent listed., Results: There were 41 400 pneumonia deaths during the study period, corresponding to an annualized age-adjusted death rate of 29.7 per 100 000 population. Approximately 17.5% of pneumonia deaths specified an etiologic agent. Age-adjusted pneumonia death rate declined over the study period and across each borough. Males had an annualized age-adjusted pneumonia death rate 1.5 (95% confidence interval [CI], 1.5-1.5) times that of females. Non-Hispanic blacks had an annualized age-adjusted pneumonia death rate 1.2 (95% CI, 1.2-1.2) times that of non-Hispanic whites. The annualized pneumonia death rate increased with age group above 5-24 years and neighborhood-level poverty. Staten Island had an annualized age-adjusted pneumonia death rate 1.3 (95% CI, 1.2-1.3) times that of Manhattan. In the multivariable analysis, pneumonia deaths were more likely to occur among males, non-Hispanic blacks, persons aged ≥65 years, residents of neighborhoods with higher poverty levels, and in Staten Island., Conclusions: While the accuracy of death certificates is unknown, investigation is needed to understand why certain populations are disproportionately recorded as dying from pneumonia in NYC.

Deaths attributable to hepatitis C (HCV) infection are increasing in the USA even as highly effective treatments become available. Neighborhood-level inequalities create barriers to care and treatment for many vulnerable populations. We seek to characterize citywide trends in HCV mortality rates over time and identify and describe neighborhoods in New York City (NYC) with disproportionately high rates and associated factors. We used a multiple cause of death (MCOD) definition for HCV mortality. Cases identified between January 1, 2006, and December 31, 2014, were geocoded to NYC census tracts (CT). We calculated age-adjusted HCV mortality rates and identified spatial clustering using a local Moran's I test. Temporal trends were analyzed using joinpoint regression. A multistep global and local Poisson modeling approach was used to test for neighborhood associations with sociodemographic indicators. During the study period, 3697 HCV-related deaths occurred in NYC, with an average annual percent increase of 2.6% (p = 0.02). The HCV mortality rates ranged from 0 to 373.6 per 100,000 by CT, and cluster analysis identified significant clustering of HCV mortality (I = 0.23). Regression identified positive associations between HCV mortality and the proportion of non-Hispanic black or Hispanic residents, neighborhood poverty, education, and non-English-speaking households. Local regression estimates identified spatially varying patterns in these associations. The rates of HCV mortality in NYC are increasing and vary by neighborhood. HCV mortality is associated with many indicators of geographic inequality. Results identified neighborhoods in greatest need for place-based interventions to address social determinants that may perpetuate inequalities in HCV mortality.

The C. elegans gut descends from the E progenitor cell through a series of stereotyped cell divisions and morphogenetic events. Effects of perturbations of upstream cell specification on downstream organogenesis have not been extensively investigated. Here we have assembled an allelic series of strains that variably compromise specification of E by perturbing the activation of the gut-specifying end-1 and end-3 genes. Using a marker that allows identification of all E descendants regardless of fate, superimposed with markers that identify cells that have adopted a gut fate, we have examined the fate of E lineage descendants among hundreds of embryos. We find that when specification is partially compromised, the E lineage undergoes hyperplasia accompanied by stochastic and variable specification of gut fate among the E descendants. As anticipated by prior work, the activation of the gut differentiation factor elt-2 becomes delayed in these strains, although ultimate protein levels of a translational ELT-2::GFP reporter resemble those of the wild type. By comparing these effects among the various specification mutants, we find that the stronger the defect in specification (i.e. the fewer number of embryos specifying gut), the stronger the defects in the E lineage and delay in activation of elt-2. Despite the changes in the E lineage in these strains, we find that supernumerary E descendants that adopt a gut fate are accommodated into a relatively normal-looking intestine. Hence, upstream perturbation of specification dramatically affects the E lineage, but as long as sufficient descendants adopt a gut fate, organogenesis overcomes these effects to form a relatively normal intestine., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1 , three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans , suggesting an analogous role for DRH-1 as an intracellular dsRNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral siRNAs in C. elegans As mammals produce Dicer-dependent viral siRNAs to target RNA viruses, our findings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs., (Copyright © 2017 Coffman et al.)

Objectives: We evaluated use of the Index of Concentration at the Extremes (ICE) for public health monitoring., Methods: We used New York City data centered around 2010 to assess cross-sectional associations at the census tract and community district levels, for (1) diverse ICE measures plus the US poverty rate, with (2) infant mortality, premature mortality (before age 65 years), and diabetes mortality., Results: Point estimates for rate ratios were consistently greatest for the novel ICE that jointly measured extreme concentrations of income and race/ethnicity. For example, the census tract-level rate ratio for infant mortality comparing the bottom versus top quintile for an ICE contrasting low-income Black versus high-income White equaled 2.93 (95% confidence interval [CI] = 2.11, 4.09), but was 2.19 (95% CI = 1.59, 3.02) for low versus high income, 2.77 (95% CI = 2.02, 3.81) for Black versus White, and 1.56 (95% CI = 1.19, 2.04) for census tracts with greater than or equal to 30% versus less than 10% below poverty., Conclusions: The ICE may be a useful metric for public health monitoring, as it simultaneously captures extremes of privilege and deprivation and can jointly measure economic and racial/ethnic segregation.

The MED-1,2 GATA factors contribute to specification of E, the progenitor of the Caenorhabditis elegans endoderm, through the genes end-1 and end-3, and in parallel with the maternal factors SKN-1, POP-1 and PAL-1. END-1,3 activate elt-2 and elt-7 to initiate a program of intestinal development, which is maintained by positive autoregulation. Here, we advance the understanding of MED-1,2 in E specification. We find that expression of end-1 and end-3 is greatly reduced in med-1,2(-) embryos. We generated strains in which MED sites have been mutated in end-1 and end-3. Without MED input, gut specification relies primarily on POP-1 and PAL-1. 25% of embryos fail to make intestine, while those that do display abnormal numbers of gut cells due to a delayed and stochastic acquisition of intestine fate. Surviving adults exhibit phenotypes consistent with a primary defect in the intestine. Our results establish that MED-1,2 provide robustness to endoderm specification through end-1 and end-3, and reveal that gut differentiation may be more directly linked to specification than previously appreciated. The results argue against an "all-or-none" description of cell specification, and suggest that activation of tissue-specific master regulators, even when expression of these is maintained by positive autoregulation, does not guarantee proper function of differentiated cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: The quality of cause-of-death reporting on death certificates affects the usefulness of vital statistics for public health action. Heart disease deaths are overreported in the United States. We evaluated the impact of an intervention to reduce heart disease overreporting on other leading causes of death., Methods: A multicomponent intervention comprising training and communication with hospital staff was implemented during July through December 2009 at 8 New York City hospitals reporting excessive heart disease deaths. We compared crude, age-adjusted, and race/ethnicity-adjusted proportions of leading, underlying causes of death reported during death certification by intervention and nonintervention hospitals during preintervention (January-June 2009) and postintervention (January-June 2010) periods. We also examined trends in leading causes of death for 2000 through 2010., Results: At intervention hospitals, heart disease deaths declined by 54% postintervention; other leading causes of death (ie, malignant neoplasms, influenza and pneumonia, cerebrovascular disease, and chronic lower respiratory diseases) increased by 48% to 232%. Leading causes of death at nonintervention hospitals changed by 6% or less. In the preintervention period, differences in leading causes of death between intervention and nonintervention hospitals persisted after controlling for race/ethnicity and age; in the postintervention period, age accounted for most differences observed between intervention and nonintervention hospitals. Postintervention, malignant neoplasms became the leading cause of premature death (ie, deaths among patients aged 35-74 y) at intervention hospitals., Conclusion: A hospital-level intervention to reduce heart disease overreporting led to substantial changes to other leading causes of death, changing the leading cause of premature death. Heart disease overreporting is likely obscuring the true levels of cause-specific mortality.

Objectives: Heart disease death overreporting is problematic in New York City (NYC) and other US jurisdictions. We examined whether overreporting affects the premature (< 65 years) heart disease death rate disparity between non-Hispanic Blacks and non-Hispanic Whites in NYC., Methods: We identified overreporting hospitals and used counts of premature heart disease deaths at reference hospitals to estimate corrected counts. We then corrected citywide, age-adjusted premature heart disease death rates among Blacks and Whites and a White-Black premature heart disease death disparity., Results: At overreporting hospitals, 51% of the decedents were White compared with 25% at reference hospitals. Correcting the heart disease death counts at overreporting hospitals decreased the age-adjusted premature heart disease death rate 10.1% (from 41.5 to 37.3 per 100,000) among Whites compared with 4.2% (from 66.2 to 63.4 per 100,000) among Blacks. Correction increased the White-Black disparity 6.1% (from 24.6 to 26.1 per 100,000)., Conclusions: In 2008, NYC's White-Black premature heart disease death disparity was underestimated because of overreporting by hospitals serving larger proportions of Whites. Efforts to reduce overreporting may increase the observed disparity, potentially obscuring any programmatic or policy-driven advances.

Few studies have investigated heterogeneity of selection response in replicate lines subjected to equivalent selection. We developed four replicate lines of mice based on high levels of voluntary wheel running (high runner or HR lines) while also maintaining four nonselected control lines. This led to the unexpected discovery of the HR minimuscle (HRmini) phenotype, recognized by a 50% reduction in hindlimb muscle mass, which became fixed in 1 of the four HR selected lines. Here, we report genome-wide expression profiling describing transcriptome differences between HRnormal and HRmini medial gastrocnemius. Consistent with the known reduction of type IIB fibers in HRmini, Myh4 gene expression was -8.82-fold less (P = 0.0001) in HRmini, which was closely associated with differences in the "calcium signaling" canonical pathway, including structural genes (e.g., Mef2c, twofold greater in HRmini, P = 0.0003) and myogenic factors (e.g., Myog, 3.8-fold greater in HRmini, P = 0.0026) associated with slow-type myofibers. The gene that determines the HRmini phenotype is known to reside in a 2.6335-Mb interval on mouse chromosome 11 and 7 genes (Myh10, Chrnb1, Acadvl, Senp3, Gabarap, Eif5a, and Clec10a) from this region were differentially expressed. Verification by real-time PCR confirmed 1.5-fold greater (P < 0.05) expression of very long chain acyl-CoA dehydrogenase (Acadvl) in HRmini. Ten other genes associated with fatty acid metabolism were also upregulated in HRmini, suggesting differences in the ability to metabolize fatty acids in HRnormal and HRmini muscles. This work provides a resource for understanding differences in muscle phenotypes in populations exhibiting high running capacity.

Introduction: Poor-quality cause-of-death reporting reduces reliability of mortality statistics used to direct public health efforts. Overreporting of heart disease has been documented in New York City (NYC) and nationwide. Our objective was to evaluate the immediate and longer-term effects of a cause-of-death (COD) educational program that NYC's health department conducted at 8 hospitals on heart disease reporting and on average conditions per certificate, which are indicators of the quality of COD reporting., Methods: From June 2009 through January 2010, we intervened at 8 hospitals that overreported heart disease deaths in 2008. We shared hospital-specific data on COD reporting, held conference calls with key hospital staff, and conducted in-service training. For deaths reported from January 2009 through June 2011, we compared the proportion of heart disease deaths and average number of conditions per death certificate before and after the intervention at both intervention and nonintervention hospitals., Results: At intervention hospitals, the proportion of death certificates that reported heart disease as the cause of death decreased from 68.8% preintervention to 32.4% postintervention (P < .001). Individual hospital proportions ranged from 58.9% to 79.5% preintervention and 25.9% to 45.0% postintervention. At intervention hospitals the average number of conditions per death certificate increased from 2.4 conditions preintervention to 3.4 conditions postintervention (P < .001) and remained at 3.4 conditions a year later. At nonintervention hospitals, these measures remained relatively consistent across the intervention and postintervention period., Conclusion: This NYC health department's hospital-level intervention led to durable changes in COD reporting.

Matrotrophic fish in the genus Poeciliopsis (Poeciliidae) have a placenta-like structure used in postfertilization maternal provisioning of the developing embryo. To understand better the structure and function of the Poeciliopsis placenta, we derived cDNA libraries from the maternal follicular placenta of 2 matrotrophic Poeciliopsis sister species, P. turneri and P. presidionis. These species inherited their placenta from a common ancestor and represent one of 3 independent origins of placentas in Poeciliopsis. Expressed sequence tags (ESTs) were generated and putative function was determined using BLASTX homology searches and Gene Ontology (GO) annotation. Reverse transcription-polymerase chain reaction was used to verify placenta tissue expression of a putative candidate gene, alpha-2 macroglobulin. In total, 1956 (71.5% of the total submitted ESTs) and 924 (71.0% of the total submitted ESTs) unique transcripts were identified for the P. turneri and P. presidionis placenta, respectively. Homology search and GO annotation revealed putative genes whose products may be involved in specific transport functions of the maternal follicle. These putative genes are excellent candidates for future research on the evolution of the placenta. We discuss our results in light of the parent-offspring conflict theory of placental evolution and in terms of the Poeciliid placenta structure and function.

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.

In C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/beta-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the mis-specification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, a previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35(-) embryonic arrest phenotype was variable. We report here that the NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and that CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. Our data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, our results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm.

MED-1 is a member of a group of divergent GATA-type zinc finger proteins recently identified in several species of Caenorhabditis. The med genes are transcriptional regulators that are involved in the specification of the mesoderm and endoderm precursor cells in nematodes. Unlike other GATA-type zinc fingers that recognize the consensus sequence (A/C/T)GATA(A/G), the MED-1 zinc finger (MED1zf) binds the larger and atypical site GTATACT(T/C)(3). We have examined the basis for this unusual DNA specificity using a range of biochemical and biophysical approaches. Most strikingly, we show that although the core of the MED1zf structure is similar to that of GATA-1, the basic tail C-terminal to the zinc finger unexpectedly adopts an alpha-helical structure upon binding DNA. This additional helix appears to contact the major groove of the DNA, making contacts that explain the extended DNA consensus sequence observed for MED1zf. Our data expand the versatility of DNA recognition by GATA-type zinc fingers and perhaps shed new light on the DNA-binding properties of mammalian GATA factors.

In the nematode, C. elegans, the bZIP/homeodomain transcription factor SKN-1 and the Wnt effector TCF/POP-1 are central to the maternal specification of the endomesoderm prior to gastrulation. The 8-cell stage blastomere MS is primarily a mesodermal precursor, giving rise to cells of the pharynx and body muscle among others, while its sister E clonally generates the entire endoderm (gut). In C. elegans, loss of SKN-1 results in the absence of MS-derived tissues all of the time, and loss of gut most of the time, while loss of POP-1 results in a mis-specification of MS as an E-like cell, resulting in ectopic gut. We show that in C. briggsae, RNAi of skn-1 results in a stronger E defect but no apparent MS defect, while RNAi of pop-1 results in loss of gut and an apparent E to MS transformation, the opposite of the pop-1 knockdown phenotype seen in C. elegans. The difference in pop-1(-) phenotypes correlates with changes in how the endogenous endoderm-specifying end genes are regulated by POP-1 in the two species. Our results suggest that integration of Wnt-dependent and Wnt-independent cell fate specification pathways within the Caenorhabditis genus can occur in different ways.

Risk adjustment is a critical tool in public reporting of quality measures. Its aim is to level the playing field so that providers serving different patients can be meaningfully compared. We used a theory and evidence-based approach to develop risk-adjustment models for the 10 publicly reported home health quality measures and compared their performance with current models developed using a data-driven stepwise approach. Overall, the quality ratings for most agencies were similar regardless of approach. Theory and evidence-based models have the potential to simplify risk adjustment, and thereby improve provider and consumer understanding and confidence in public reporting.

We have previously shown that the MED-1,2 divergent GATA factors act apparently zygotically to specify the fates of the MS (mesoderm) and E (endoderm) sister cells, born at the 7-cell stage of C. elegans embryogenesis. In the E cell, MED-1,2 activate transcription of the endoderm-promoting end-1 and end-3 genes. We demonstrate by in situ hybridization that med transcripts accumulate both in the EMS cell and in the maternal germline in a SKN-1-dependent manner. Removal of zygotic med function alone results in a weakly impenetrant loss of endoderm. However, med-1,2(-) embryos made by mothers in which germline med transcripts have been abrogated by transgene cosuppression fail to make endoderm 50% of the time, similar to the phenotype seen by RNAi. We also find that reduction of Med or End activity results in aberrant numbers of intestinal cells in embryos that make endoderm. We further show that regulation of the paralogous end-1 and end-3 genes consists of both shared and distinct inputs, and that END-3 activates end-1 expression. Our data thus reveal three new properties of C. elegans endoderm specification: both maternal and zygotic activities of the med genes act to specify endoderm, defects in endoderm specification also result in defects in gut cell number, and activation of the paralogous end-1 and end-3 genes differs qualitatively in the relative contributions of their upstream regulators.

In C. elegans, many mesodermal cell types are made by descendants of the progenitor MS, born at the seven-cell stage of embryonic development. Descendants of MS contribute to body wall muscle and to the posterior half of the pharynx. We have previously shown that MS is specified by the activity of the divergent MED-1,2 GATA factors. We report that the MED-1,2 target gene tbx-35, which encodes a T-box transcription factor, specifies the MS fate. Embryos homozygous for a putative tbx-35-null mutation fail to generate MS-derived pharynx and body muscle, and instead generate ectopic PAL-1-dependent muscle and hypodermis, tissues normally made by the C blastomere. Conversely, overexpression of tbx-35 results in the generation of ectopic pharynx and muscle tissue. The MS and E sister cells are made different by transduction of a Wnt/MAPK/Src pathway signal through the nuclear effector TCF/POP-1. We show that in E, tbx-35 is repressed in a Wnt-dependent manner that does not require activity of TCF/POP-1, suggesting that an additional nuclear Wnt effector functions in E to repress MS development. Genes of the T-box family are known to function in protostomes and deuterostomes in the specification of mesodermal fates. Our results show that this role has been evolutionarily conserved in the early C. elegans embryo, and that a progenitor of multiple tissue types can be specified by a surprisingly simple gene cascade.

Mesoderm and endoderm in C. elegans arise from sister cells called MS and E, respectively. The identities of both of these mesendodermal progenitors are controlled by MED-1 and -2, members of the GATA factor family. In the E lineage, these factors activate a sequential cascade of GATA factors, beginning with their immediate targets, the endoderm-specifying end genes. We report that MED-1 binds invariant noncanonical sites in the end genes, revealing that the MEDs are atypical members of the GATA factor family that do not recognize GATA sequences. By searching the genome for clusters of these MED sites, we have identified 19 candidate MED targets. Based on their expression patterns, these define three distinct classes of MED-regulated genes: MS-specific, E-specific, and E plus MS-specific. Some MED targets encode transcription factors related to those that regulate mesendoderm development in other phyla, supporting the existence of an ancient metazoan mesendoderm gene regulatory network.

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.