Circadian activation of the mitogen-activated protein kinase MAK-1 facilitates rhythms in clock-controlled genes in Neurospora crassa.

The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus Neurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in Saccharomyces cerevisiae and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from Neurospora cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ~500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e., ccg-1 and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.

A cis regulatory element in the TAPNAC promoter directs tapetal gene expression.

The tapetum is a single cell layer surrounding the anther locule and its major function is to provide nutrients for pollen development. The ablation of tapetal cells interferes with pollen production and results in plant male sterility. In spite of the importance of this tissue in the quality and production of pollen grains, studies on promoter gene regulation of tapetal expressed genes are very few and there are no reports on specific cis regulatory sequences that control tapetal gene expression. We have identified a NAC gene, TAPNAC (At1g61110), specifically expressed in the Arabidopsis tapetum via transcriptional profiling. The TAPNAC promoter was studied in detail to identify cis regulatory sequences that confer tapetal specific expression. For this purpose, TAPNAC promoter elements were fused to the ß-glucuronidase (GUS) reporter gene, and spatial and temporal GUS expression was monitored. The results showed that TAPNAC promoter-driven GUS expression emulates the expression of TAPNAC mRNA in anthers. A conserved TCGTGT motif was identified in the TAPNAC promoter and other tapetal expressed promoters. The TCGTGT motif enhances GUS expression in anthers of transgenic plants but only in the context of the TAPNAC promoter proximal region.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

Sugarcane DIRIGENT and O-methyltransferase promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Omicron-Methyltransferase (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the beta-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both Pro( DIR16 ):GUS and Pro( OMT ):GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. Pro( DIR16 ) and Pro( OMT ) will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Isolating promoters of multigene family members from the polyploid sugarcane genome by PCR-based walking in BAC DNA.

The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.

Circadian genomics of the chick pineal gland in vitro.

BACKGROUND: Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. These properties make the in vitro pineal a particularly useful model for exploring circadian control of gene transcription in a pacemaker tissue, as well as regulation of the transcriptome by primary inputs to the clock (both photic and noradrenergic). RESULTS: We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD. We report that a reduced subset of genes was rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially enhanced or suppressed specific sets of genes in a fashion that was dependent upon time of day. CONCLUSION: Our combined approach of utilizing a temporal, photic and pharmacological microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in circadian physiology. Further, we hypothesize that the pineal circadian transcriptome is reduced but functionally conserved in vitro, and supports an endogenous role for the pineal in regulating local rhythms in metabolism, immune function, and other conserved pathways.

Circadian profiling of the transcriptome in NIH/3T3 fibroblasts: comparison with rhythmic gene expression in SCN2.2 cells and the rat SCN.

To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Cry1, and Bmal1, and 323 functionally diverse transcripts, many of which regulate cellular communication. Overlap in rhythmic transcripts among NIH/3T3 fibroblasts, SCN2.2 cells, and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared with NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (iNos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in cocultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. This comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.

Transcriptional profiling of the chick pineal gland, a photoreceptive circadian oscillator and pacemaker.

The avian pineal gland contains both circadian oscillators and photoreceptors to produce rhythms in biosynthesis of the hormone melatonin in vivo and in vitro. The molecular mechanisms for melatonin biosynthesis are largely understood, but the mechanisms driving the rhythm itself or the photoreceptive processes that entrain the rhythm are unknown. We have produced cDNA microarrays of pineal gland transcripts under light-dark and constant darkness conditions. Rhythmic transcripts were classified according to function, representing diverse functional groups, including phototransduction pathways, transcription/translation factors, ion channel proteins, cell signaling molecules, and immune function genes. These were also organized relative to time of day mRNA abundance in light-dark and constant darkness. The transcriptional profile of the chick pineal gland reveals a more complex form of gene regulation than one might expect from a gland whose sole apparent function is the rhythmic biosynthesis of melatonin. The mRNAs encoding melatonin biosynthesis are rhythmic as are many orthologs of mammalian "clock genes." However, the oscillation of phototransductive, immune, stress response, hormone binding, and other important processes in the transcriptome of the pineal gland, raises new questions regarding the role of the pineal gland in circadian rhythm generation, organization, and avian physiology.

Helicobacter pylori infection in rural China: demographic, lifestyle and environmental factors.

BACKGROUND: Although Helicobacter pylori is one of the most common human bacterial infections worldwide, its mode of transmission is unclear. METHODS: To investigate possible associations between H. pylori infection and demographic, lifestyle, and environmental factors in a rural Chinese population, a cross-sectional survey was administered to 3288 adults (1994 seropositive, 1019 seronegative, 275 indeterminate) from 13 villages in Linqu County, Shandong Province, China. RESULTS: Helicobacter pylori prevalence was elevated for: infrequent handwashing before meals (OR = 1.7, 95% CI: 1.0-3.0), crowding (i.e. sharing a bed with >2 people [OR = 2.3, 95% CI: 1.3-4.2]), washing/bathing in a pond or ditch (OR = 1.5, 95% CI: 1.0-2.4), and medium (OR = 1.6, 95% CI: 1.3-2.0) and low (OR = 2.3, 95% CI: 1.9-2.9) compared to high village education level, and reduced for never being married or divorced (OR = 0.4, 95% CI: 0.2-1.0). There was also a suggestion that source of drinking water, especially water from a shallow village well might be related to H. pylori seropositivity. There was no evidence of an association between H. pylori prevalence and alcohol or tobacco use, raw fruit and vegetable intake, or individual social class measures. CONCLUSIONS: The results of this study suggest that person-to-person transmission is the most plausible route of H. pylori infection in this rural Chinese population, but waterborne exposures deserve further investigation.

A cohort study of thyroid cancer and other thyroid diseases after the Chornobyl accident: objectives, design and methods.

The thyroid gland in children is one of the organs that is most sensitive to external exposure to X and gamma rays. However, data on the risk of thyroid cancer in children after exposure to radioactive iodines are sparse. The Chornobyl accident in Ukraine in 1986 led to the exposure of large populations to radioactive iodines, particularly (131)I. This paper describes an ongoing cohort study being conducted in Belarus and Ukraine that includes 25,161 subjects under the age of 18 years in 1986 who are being screened for thyroid diseases every 2 years. Individual thyroid doses are being estimated for all study subjects based on measurement of the radioactivity of the thyroid gland made in 1986 together with a radioecological model and interview data. Approximately 100 histologically confirmed thyroid cancers were detected as a consequence of the first round of screening. The data will enable fitting appropriate dose-response models, which are important in both radiation epidemiology and public health for prediction of risks from exposure to radioactive iodines from medical sources and any future nuclear accidents. Plans are to continue to follow-up the cohort for at least three screening cycles, which will lead to more precise estimates of risk.

Health experience of 122 submarine crewmembers during a 101-day submergence.

INTRODUCTION: The artificial environment encountered in submarine duty may affect the health of crewmembers. Previous studies of submariner health have not examined self-treatment habits or the incidence of minor health problems for which no care is sought from a health care provider. Our study examined patterns of minor medical problems and self-treatment among the crew of one submarine over a period of continued submergence for 101 d. METHODS: Study data were obtained from three self-administered serial surveys of 122 medically screened U.S. Navy personnel onboard a submerged nuclear-powered submarine at the beginning, middle, and end of the study. RESULTS: During the first half of the study, 82% had medical complaints, most commonly runny nose, difficulty sleeping, and backache. In the second half, 77% listed complaints, most commonly difficulty sleeping. Despite readily available medical care, self-medication for minor unreported health problems was common, with use of products such as non-steroidal anti-inflammatory drugs, multi-vitamins, health supplements, topical preparations, and antihistamine/decongestants. DISCUSSION: Planning for medical care in isolated environments should include consideration of inreported minor medical problems and self-treatment patterns.

Health of U.S. Navy submarine crew during periods of isolation.

BACKGROUND: An essential element in planning for long-term space missions is prediction of the medical support required. Medical data for analogous populations serving in isolated and/or contained environments are useful in predicting health risks for astronauts. METHODS: This study evaluated the rates of health events that occurred among a highly screened, healthy military population during periods of isolation using a centralized database of medical encounter records from U.S. Navy submarines. The study population was composed of U.S. Navy officers and enlisted men deployed on 240 submarine patrols between 1 January 1997 and 30 September 2000. RESULTS: A total of 1389 officers and 11,952 enlisted crew members served aboard participating submarines for 215,086 and 1,955,521 person-days at sea, respectively, during the study period. Officers had 214 initial visits to medical staff with 79 re-visits for the same condition during these patrols, while enlisted men had 3345 initial visits and 1549 re-visits. Among officers, the most common category of medical events was respiratory illnesses (primarily upper respiratory infections), followed by injury, musculoskeletal conditions, infectious diseases, symptoms and ill-defined conditions, and skin problems. Among enlisted men, the most common category of medical events was injury, followed by respiratory illnesses (upper respiratory infections), skin problems, symptoms and ill-defined conditions, digestive disorders, infectious conditions, sensory organ problems (ear infections and eye problems), and musculoskeletal conditions. CONCLUSIONS: Potential mission-impacting medical events reported were rare, i.e., among a crew of seven officers, only one medical event would be expected to occur during a 6-mo mission and result in 3/4 d or less of limited or no duty. Among a crew of seven enlisted men, about two medical events would be expected during a 6-mo mission and result in about 1 d of limited or no duty per medical event.

Reproducible RNA preparation from sugarcane and citrus for functional genomic applications.

High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and citrus, which provide a challenge due to the presence of fiber, polysaccharides, or secondary metabolites. The RNA isolated by this method is suitable for several downstream applications including northern blot hybridization, microarray analysis, and quantitative RT-PCR. This method has been used in a diverse range of projects ranging from screening plant lines overexpressing mammalian genes to analyzing plant responses to viral infection and defense signaling molecules.

Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice.

The virB operon, encoding a Type IV secretion system (T4SS), is essential for intracellular survival and persistent infection by Brucella spp. To better understand the role of the T4SS in evading host defence mechanisms and establishing chronic infection, we compared transcriptional profiles of the host response to infection with wild-type and virB mutant Brucella strains. Analysis of gene expression profiles in murine splenocytes 3 days after inoculation with wild-type Brucella strains revealed an inflammatory response, with a prominent upregulation of genes induced by both type I and type II interferons. Real-time RT-PCR showed that a group of genes from these pathways were induced by day 3 post infection and declined to baseline levels by day 7. In contrast, neither of the two virB mutant strains elicited a proinflammatory gene expression profile, demonstrating that the T4SS was required to trigger this response. Infection studies using type I interferon receptor knockout mice showed that a lack of type I interferon signalling did not affect Brucella replication during the first 4 weeks of infection. Thus, induction of type I interferons does not appear to be an essential mechanism by which the T4SS promotes persistent infection by Brucella.

Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus.

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

A cohort study of thyroid cancer and other thyroid diseases after the chornobyl accident: thyroid cancer in Ukraine detected during first screening.

BACKGROUND: The Chornobyl accident in 1986 exposed thousands of people to radioactive iodine isotopes, particularly (131)I; this exposure was followed by a large increase in thyroid cancer among those exposed as children and adolescents, particularly in Belarus, the Russian Federation, and Ukraine. Here we report the results of the first cohort study of thyroid cancer among those exposed as children and adolescents following the Chornobyl accident. METHODS: A cohort of 32 385 individuals younger than 18 years of age and resident in the most heavily contaminated areas in Ukraine at the time of the accident was invited to be screened for any thyroid pathology by ultrasound and palpation between 1998 and 2000; 13 127 individuals (44%) were actually screened. Individual estimates of radiation dose to the thyroid were available for all screenees based on radioactivity measurements made shortly after the accident and on interview data. The excess relative risk per gray (Gy) was estimated using individual doses and a linear excess relative risk model. RESULTS: Forty-five pathologically confirmed cases of thyroid cancer were found during the 1998-2000 screening. Thyroid cancer showed a strong, monotonic, and approximately linear relationship with individual thyroid dose estimate (P<.001), yielding an estimated excess relative risk of 5.25 per Gy (95% confidence interval [CI] = 1.70 to 27.5). Greater age at exposure was associated with decreased risk of radiation-related thyroid cancer, although this interaction effect was not statistically significant. CONCLUSION: Exposure to radioactive iodine was strongly associated with increased risk of thyroid cancer among those exposed as children and adolescents. In the absence of Chornobyl radiation, 11.2 thyroid cancer cases would have been expected compared with the 45 observed, i.e., a reduction of 75% (95% CI = 50% to 93%). The study also provides quantitative risk estimates minimally confounded by any screening effects. Caution should be exercised in generalizing these results to any future similar accidents because of the potential differences in the nature of the radioactive iodines involved, the duration and temporal patterns of exposures, and the susceptibility of the exposed population.

Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot Dc3 gene.

The carrot (Daucus carota L.) lea-class gene Dc3 is expressed in developing seeds and in vegetative tissues subject to drought and treatment with exogenous abscisic acid (ABA). Cis regulatory elements involved in seed-specific expression and in response to ABA were identified in transgenic tobacco (Nicotiana tabacum L.) using beta-glucuronidase (GUS) reporter gene constructs containing a series of deletion and orientation mutants of the Dc3 promoter. These experiments demonstrated that the Dc3 promoter is comprised of a proximal promoter region (PPR) and a distal promoter region (DPR). TCGTGT motifs in the DPR in combination with the PPR comprise a novel, bipartite ABA module in the Dc3 gene promoter. The PPR contains cis-acting elements responsible for the developmental regulation of Dc3 expression in seeds. Five similar sequence motifs with the consensus ACACgtGCa were identified in the PPR. Both DPR and PPR interact with common nuclear proteins that are present in embryos and are inducible by ABA in vegetative tissues.

Redundant and distinct functions of the ABA response loci ABA-INSENSITIVE(ABI)5 and ABRE-BINDING FACTOR (ABF)3.

Abscisic acid-responsive gene expression is regulated by numerous transcription factors, including a subgroup of basic leucine zipper factors that bind to the conserved cis-acting sequences known as ABA-responsive elements. Although one of these factors, ABA-insensitive 5 (ABI5), was identified genetically, the paucity of genetic data for the other family members has left it unclear whether they perform unique functions or act redundantly to ABI5 or each other. To test for potential redundancy with ABI5, we identified the family members with most similar effects and interactions in transient expression systems (ABF3 and ABF1), then characterized loss-of-function lines for those loci. The abf1 and abf3 monogenic mutant lines had at most minimal effects on germination or seed-specific gene expression, but the enhanced ABA- and stress-resistance of abf3 abi5 double mutants revealed redundant action of these genes in multiple stress responses of seeds and seedlings. Although ABI5, ABF3, and ABF1 have some overlapping effects, they appear to antagonistically regulate each other's expression at specific stages. Consequently, loss of any one factor may be partially compensated by increased expression of other family members.

A gene network downstream of transcription factor Math5 regulates retinal progenitor cell competence and ganglion cell fate.

Math5, a mouse homolog of the Drosophila proneural bHLH transcription factor Atonal, is essential in the developing retina to establish retinal progenitor cell competence for a ganglion cell fate. Elucidating the mechanisms by which Math5 influences progenitor cell competence is crucial for understanding how specification of neuronal cell fate occurs in the retina and it requires knowledge of the downstream target genes that depend on Math5 for their expression. To date, only a handful of genes downstream of Math5 have been identified. To better define the gene network operating downstream of Math5, we used custom-designed microarrays to examine the changes in embryonic retinal gene expression caused by deletion of math5. We identified 270 Math5-dependent genes, including those that were expressed specifically either in progenitor cells or differentiated ganglion cells. The ganglion cell-specific genes included both Brn3b-dependent and Brn3b-independent genes, indicating that Math5 regulates distinct branches of the gene network responsible for retinal ganglion cell differentiation. In math5-null progenitor cells, there was an up-regulation of the proneural genes math3, neuroD, and ngn2, indicating that Math5 suppresses the production of other cell types in addition to promoting retinal ganglion cell formation. The promoter regions of many Math5-dependent genes contained binding sites for REST/NRSF, suggesting that release from general repression in retinal progenitor cells is required for ganglion cell-specific gene activation. The identification of multiple roles for Math5 provides new insights into the gene network that defines progenitor cell competence in the embryonic retina.