Maduro, Morris et al. (2019) International Worm Meeting "Modulation of metabolism by FSTR-1/F57F4.3 and FSTR-2/F57F4.4."

Dimov, Ivan, Maduro, Morris, Rivera, Madeline, Soto, Jose, Broitman-Maduro, Gina, Choi, Hailey

[International Worm Meeting, 2019]

Organisms modulate metabolic pathways dependent on life stage, nutrition, and environment, among other factors. CLK-1, an ortholog of human COQ7, is necessary for ubiquinone biosynthesis (Liu et al., 2017, PMID: 28404998). Mutation of clk-1 results in many pleiotropic effects, including a slowing down of metabolic and rhythmic processes. The paralogous fstr-1 and fstr-2 genes were identified by the ability of their downregulation by RNAi to restore the speed of rhythmic behaviors that are slowed in clk-1 mutants (Cristina et al., 2009, PMID: 19360127). FSTR-1/F57F4.3 and FSTR-2/F57F4.4 are paralogous proteins that are 96% identical and differ primarily by the absence of a small 63-aa region in FSTR-2. Both proteins are each >2000 amino acids long and contain many repeated ET domains of unknown function. We have previously found that strains in which gut specification is partially compromised ('hypomorphic gut specification' or HGS strains), animals accumulate extra lipids (Maduro et al., 2015, PMID: 25959238). Through RNA-seq analysis of intestine-enriched genes, we found that in wild-type strains, fstr-2/F57F4.4 is upregulated, while in HGS strains, fstr-1/F57F4.3 is upregulated. We hypothesized, therefore, that FSTR-1 and FSTR-2 play related but differentiable roles in regulating metabolism. Consistent with a focus in the intestine, wrmScarlet reporters (transcriptional and translational) are enriched in the gut, with primarily cytoplasmic localization. To further characterize the roles of fstr-1 and fstr-2 in metabolism, we generated single and double knockout alleles by CRISPR/Cas9-mediated mutagenesis. Both fstr-1 fstr-2 double mutants and fstr-2 single mutants appear to contain more gut granules and have a different distribution of lipids than controls, suggesting that FSTR-2 has a role in regulation of normal metabolism in the intestine. While we find no evidence of a changed life span in fstr-1 fstr-2 double mutants, these animals exhibit increased susceptibility to toxin-mediated killing by Pseudomonas aeruginosa. Others have reported upregulation of fstr-2::GFP during pathogen infection (Julien-Gau et al., 2014, PMID: 24012871). As a further means of measuring possible changes in metabolism in fstr mutants and our original HGS strains, we have introduced a gut-expressed ATP FRET sensor (Mendelsohn et al., 2018, PMID: 30148842) into these backgrounds. Our preliminary data suggest that both clk-1 and fstr-1 fstr-2 double mutants have at most a mild impairment in ATP production in the intestine, at least in the first few days of adulthood. These results are consistent with biochemical studies on clk-1 mutants (Braeckman et al., 1999, PMID: 10330373). Overall, our results support a role for FSTR-2 in modulation of metabolism under some conditions. Experiments to probe differences between FSTR-1 and FSTR-2 in wild-type and HGS strains are ongoing.

Maduro, Morris (2011) Worm Breeder's Gazette "Omission of RNase and addition of ethanol precipitation improve transgenesis using spin column-purified DNA"

Maduro, Morris

[Worm Breeder's Gazette, 2011]

Maduro, Morris (2019) International Worm Meeting "A majors-level Intro Bio course consisting of online videos is at least as good as a traditional class."

Maduro, Morris

[International Worm Meeting, 2019]

I have taught Introductory Cell and Molecular Biology to hundreds of students every year since 2004. Over the years, I and other instructors have turned to clickers as a primary means of engaging students in class. I have found that these make little difference in exam performance, but the more fundamental problem is that students do not learn much from lectures, and they do not like sitting in large lecture halls. Rather than try to make the classroom experience more engaging and active, I did the opposite and got rid of the lecture, converting the entire 10-week course into 75 short videos of total length ~9 hours. I made classroom attendance optional except for the exams. For the few students that attend (about 15% of the enrollment), I take questions and cover selected topics. The videos consist primarily of PowerPoint with narration, with some of me in front of the camera at a whiteboard. All videos have closed captions. There is a multiple-choice question every two minutes that students must answer; the videos are run in PlayPosit (www.playposit.com) which keeps track of student progress in real time. Students will see about 350 questions across the entire course. They also have a face-to-face discussion with a TA once per week (graded only for attendance), access to my course notes, and a collection of my past exams. The lab part of the course is handled separately from the lecture/discussion. From several offerings of this Hybrid course, students generally respond favorably to this format. Over 95% of the enrolled students complete each video on time, as compared with lecture attendance in the traditional format, which often went below 50% by mid-course. By also offering three midterms instead of two, each exam has fewer questions and covers less material. These two factors seem to account for a ~5% increase in exam performance compared with the traditional format. Teaching evaluations have shown an increase as well, which is surprising since 85% of the students have never seen me teach in person. These results confirm that today's undergraduate students can engage just as well, and possibly better, with a well-structured Hybrid intro bio course compared with a traditional lecture-based course. Similar studies have found the same results (e.g. Gavassa et al., 2018, PMID: 30807254). For some instructors of life sciences courses, it may be worth restructuring.

Maduro, Morris et al. (2021) International Worm Meeting "Opposing roles of DAF-16 and NHR-156 in regulation of metabolism downstream of gut specification"

Soto, Jose, Choi, Hailey, Maduro, Morris, Dimov, Ivan, Broitman-Maduro, Gina

[International Worm Meeting, 2021]

We previously reported that genetic perturbations that partially compromise through the gut specification factors END-1 and END-3 result in characteristic "hypomorphic gut specification" or HGS defects, apparently resulting from variability in the timely activation of ELT-2. Even though HGS embryos may be delayed in ELT-2 activation, final ELT-2 levels are comparable to normal animals. HGS defects include accumulation of fat (dyslipidemia) seen by accumulation of large fat deposits seen by Oil Red O and a fluorescent in vivo fluorescent Perilipin/PLIN-1 marker for lipid droplets. The fat defects may be part of a global change in metabolism, as an ATP sensor suggests that steady-state levels of ATP are reduced in HGS strains. Such animals also display a range of pleiotropic effects on fertility and body morphology, among others, that could be downstream of these metabolic changes. From RNA-Seq studies on isolated intestinal tissue from HGS adults, we identified a small set of differentially expressed regulators, including the Nuclear Hormone Receptor gene nhr-156. In HGS adults, nhr-156 is downregulated relative to the wild type. A deletion mutation in the gene results in a similar hyperlipidemia as seen in the most extreme of HGS adults, suggesting that a major component of the fat accumulation in HGS strains is the reduction of expression of this NHR. We find that restoring expression of NHR-156 to HGS strains results in partial rescue of the lipid defect, consistent with a model in which hypomorphic gut specification defects result from decreased expression of nhr-156 in larvae and adults. Our earlier results had also shown that accumulation and distribution of lipids in HGS adults was dependent partly on DAF-16, as simultaneous loss of DAF-16 in HGS strains caused a partial suppression of the excess of Oil Red O staining. We now find that in HGS strains, loss of DAF-16 and forced expression of NHR-156 synergistically restore fat distribution to near wild-type. The data suggest a model in which an early failure to robustly activate ELT-2 leads to a subsequent failure to activate nhr-156 to high levels, leading to changes in fat storage and metabolism that are dependent on both repression of NHR-156 and activation of DAF-16. These results connect intestinal specification with function of the differentiated gut, suggesting that robust embryonic specification of intestine is required to set a correct metabolic state. Unexpectedly, the apparently normal expression of ELT-2, even if delayed by only hours, misses some time window to set this state.

Maduro, Morris (2021) International Worm Meeting "Making online exams more secure in an introductory cell/molecular biology course by using banks of questions made with Python"

Maduro, Morris

[International Worm Meeting, 2021]

The move to remote instruction at the onset of the pandemic changed the way that instructors give and proctor exams. The availability of resources to cheat during exams has greatly compromised the ability to assess individual learning outcomes. Restricting exams to short time intervals, randomizing question/answer order with one-at-a-time presentation of questions (and no backtracking) are considered best practices to make cheating more difficult. Another accepted best practice, but more time-consuming to implement, is to make alternate versions of exam questions. When teaching became remote for a majors-level introductory cell/molecular biology course (250+ students) in spring of 2020, I began generating question pools semiautomatically by scripting. The ease of programming in Python makes this language ideal for such an application. Questions involving simple genetics and molecular biology, factual recall, and diagram labeling, are particularly amenable to generating variant questions. Exam score distributions resembled those for in-person exams, suggesting that online exams without proctoring can generate results that are comparable to in-person exams, although there are caveats. I will outline a general approach for constructing question banks through programming and give specific examples.

Maduro, Morris F. et al. (2013) International Worm Meeting "Life at the edge of robustness: Partial guts suggest that endoderm specification is not all-or-none."

Ghamsari, Farhad, Maduro, Morris F., Broitman-Maduro, Gina, Carranza, Francisco, Walia, Gurjot

[International Worm Meeting, 2013]

The C. elegans intestine is derived from the embryonic blastomere E. The paradigm for E specification is that maternal SKN-1- and POP-1-dependent input cause activation of the E-specific gut specification factors end-1 and end-3. After reaching a threshold of expression, these activate elt-2, which maintains its expression by autoregulation and drives the commitment to gut differentiation. Prior work suggests that gut specification may not be all-or-none, and that some of the descendants of E are capable of adopting a gut fate independently of others. We have created strains in which E is specified by single-copy transgene forms of end-1 and/or end-3 that are mutated for binding sites for the MED-1,2 GATA factors, themselves direct targets of SKN-1. In such strains, different embryos make variable numbers of apparently normal-sized gut cells, suggesting that specification has become subject to stochastic variations among embryos, and that commitment to a gut fate can occur later in the E lineage. Counting of embryonic elt-2 transcripts by single-molecule FISH suggests that activation of elt-2 is more graded in these strains, as opposed to an all-or-none mode as was previously reported for SKN-1-depleted embryos (Raj et al., 2010). As these effects are confined to the E lineage due to the nature of the strains constructed, we are able to evaluate adults derived from embryos in which functional guts were made. We find that such adults store lipids at significantly higher levels and display other variable pleiotropic phenotypes, suggestive of primary defects in gut function. Together, these results build a picture in which specification of gut is not an all-or-none event, and that in animals that do make an intestine, the endoderm differentiation network is not fully self-correcting for partially compromised specification. We will present these results, including the outcome of an attempt to computationally model the stochasticity of elt-2 activation.

Maduro, Morris F. et al. (2009) International Worm Meeting "The Homeodomain protein CEH-51 and the T-box factor TBX-35 share function in specification of the MS blastomere."

Maduro, Morris F., Hung, Wendy, Broitman-Maduro, Gina, Owraghi, Melissa

[International Worm Meeting, 2009]

The MS blastomere makes some 80 embryonic cells, including pharynx cells, body muscle cells and coelomocytes. The MED-1,2 divergent GATA factors are important for specification of MS, as loss of med-1,2 results in a penetrant embryonic arrest in which embryos lack MS-derived cell types (and some of which also lack intestine, which is made by the E lineage). We previously reported the identification of the T-box factor gene tbx-35, a direct target of MED-1,2, as an important MS specification gene. However, while loss of med-1,2 results in embryos that arrest before two-fold elongation, a null mutant of tbx-35 has variable arrest phenotypes. The most severely affected tbx-35 mutants arrest at 1-fold and resemble med-1,2(-) mutants, while ~15% elongate to greater than threefold and can hatch into (inviable) larvae. At 15 deg C, even more tbx-35 mutants (up to 50%) elongate and hatch, and such embryos appear to have a partially-restored posterior pharynx, which normally contains many cells made by MS. The incomplete expressivity of the tbx-35 defects strongly suggests the existence of another gene that acts in MS specification downstream of MED-1,2. We have identified the homeobox-containing gene ceh-51 as a direct target of TBX-35. There are four binding sites for TBX-35 in the ceh-51 promoter, which define a consensus sequence similar to that of Brachyury. Consistent with its being a direct target of TBX-35, ceh-51 is expressed in the early MS lineage. However, tbx-35 mutant embryos continue to express ceh-51 at a reduced level. While tbx-35 mutants have a relatively strong MS specification defect, ceh-51 mutants have milder defects in differentiation. Mutants arrest as Unc larvae with pharynx morphology defects, and loss of ceh-51 synergizes with loss of function in hlh-1 or unc-120 to produce paralyzed, arrested two-fold (Pat) embryos (see abstract by Kuntz et al.). CEH-51 appears to be a divergent NK-2 type homeoprotein, suggesting it is an activator of genes that specify tissues among MS descendants. To directly test whether CEH-51 and TBX-35 might have overlapping functions, we constructed a tbx-35; ceh-51 strain. We found that double mutant embryos arrest with a phenotype that is very similar to med-1,2(-) embryos in elongation and development of MS-derived cell types, showing that TBX-35 and CEH-51 together account for most, if not all, of MS specification downstream of MED-1,2. Our results suggest that a gene cascade consisting of MED-1,2, TBX-35 and CEH-51 specifies the MS fate. As collaboration of function of T-box and NK-2 regulators in mesoderm development is also conserved in other animals, these results suggest that similar mechanisms specify mesoderm across multiple phyla.

Morris Maduro et al. (2008) Development & Evolution Meeting "Building the endomesoderm network: New genes and evidence for additional complexities in MS and E specification"

Katy Lin, Morris Maduro, Wendy Hung, Gina Broitman-Maduro, Melissa Owraghi

[Development & Evolution Meeting, 2008]

The MS and E blastomeres are born at the 7-cell stage of development in C. elegans. E generates the gut, while MS generates cells that are primarily mesodermal, including GLP-1-independent pharynx, body muscles, and four embryonically-derived coelomocytes. We have previously reported that tbx-35, a target of the MED-1,2 factors, is important for MS specification. In tbx-35(-) embryos, MS-derived pharynx is specifically absent, and embryos appear to have PAL-1-dependent muscle and hypodermis, characteristic of C. However, tbx-35(-) embryos still make coelomocytes from the MS cell. This suggests that it is possible for MS to adopt a mixed fate, making tissues characteristic of both MS and C, and that additional factors contribute to MS fate in the absence of TBX-35. One of these factors may be pal-1, as pal-1(RNAi); tbx-35(-) embryos make coelomocytes less frequently. Our next studies with MS and E address the role of POP-1 in MS. Using a bona fide end-1 end-3 double mutant, we find that it lacks all gut as expected, but shows a surprising ability to gastrulate normally and even hatch. Using laser ablation analysis with various GFP reporters [both early and late markers of the MS lineage], it has become clear that pop-1(-); end-1,3(-) triple mutant embryos still make GLP-1-independent pharynx tissue from MS, and also sometimes from E. To our surprise, the end-1,3 doubles and pop-1(-) alone also show activation of early MS lineage reporters in the E lineage, suggesting that both POP-1 and END-1,3 act to repress aspects of MS specification in the E cell. Finally, we have been characterizing a target of TBX-35 in the early MS lineage, provisionally named dlx-1, which encodes a homeoprotein. Overexpression of DLX-1 causes ectopic muscle to be generated, as assayed by unc-120 expression and in situ detection of ectopic myo-3 transcripts. A dlx-1::GFP reporter is expressed in the early MS lineage, and ectopically in hs-tbx-35 embryos, though it is still expressed in the tbx-35(-) mutant. A shorter promoter (-187bp) drives apparently normal early MS lineage expression but becomes completely tbx-35-dependent, suggesting that dlx-1 is a target of TBX-35 and at least one other factor in the MS lineage. Using the TBX-35 binding site identified by DNaseI footprinting of the dlx-1 promoter, we have identified one other target of TBX-35, an apparent RING finger protein. We will present the results of ongoing work on these and other projects on end-1,3 and tbx-35 regulation.

Morris Maduro et al. (2008) Development & Evolution Meeting "Your Pop's a Mom: An E to MS transformation in C. briggsae pop-1(RNAi) embryos"

Morris Maduro, Serena Cervantes, Katy Lin, Gina Broitman-Maduro, Wendy Hung

[Development & Evolution Meeting, 2008]

In 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 (the Pop phenotype). We have found that in C. briggsae, RNAi of the skn-1 ortholog results in a stronger E defect but no apparent defect in production of GLP-1-independent pharynx, one of the tissues made by MS. Laser ablations confirm that MS still makes pharynx muscle in Cb-skn-1(RNAi) embryos. Second, RNAi of pop-1 results in a highly penetrant loss of gut, and both MS and E make pharynx cells - an apparent Mom phenotype. This is the opposite of the pop-1 knockdown phenotype seen in C. elegans, in which both MS and E make gut. RNAi of the divergent beta-catenin gene Cb-sys-1 gives a much more penetrant phenotype, approximately 50% gutless, suggesting that the positive contribution of POP-1 to endoderm in C. briggsae requires interaction of Wnt-signaled POP-1 with SYS-1, just as in C. elegans. By in situ hybridization, we find that the difference in pop-1(-) phenotypes correlates with changes in how the endogenous endoderm-specifying end genes are regulated in the two species: Whereas end-1 and end-3 in C. elegans are de-repressed in MS in pop-1(RNAi), mRNAs for the C. briggsae end genes (Cb-end-1, Cb-end-3.1 and Cb-end-3.2) are no longer detectable in Cb-pop-1(RNAi). One hypothesis is that a change in cis-regulation is responsible for the difference in pop-1(-) readout. To test this, we introduced a Cb-end-3.2::GFP reporter into C. elegans. While the reporter is activated in the early E lineage in a manner similar to Ce-end-1/3 reporters, pop-1(RNAi) results in de-repression of Cb-end-3.2::GFP in the MS lineage. With the caveat that we may not have included all regulatory sites, this result suggests that the POP-1 regulation differences cannot be collectively explained solely by promoter changes. As Cb-POP-1 can complement maternal loss of Ce-pop-1 in C. elegans, we hypothesize that degeneracy of the SKN-1 pathway may account for the differences we are seeing in C. briggsae. Curiously, C. remanei pop-1(RNAi) results in an apparent ectopic endoderm phenotype, further suggesting that these changes have evolved on a relatively short timescale (tens of millions of years), as C. remanei and C. briggsae are considered to be more closely related. Our results suggest that both Wnt-dependent and Wnt-independent cell fate specification pathways in Caenorhabditis embryos have an unexpected degree of flexibility.

Morris F Maduro et al. (2002) West Coast Worm Meeting "Identification of Embryonic Mesoderm Genes Using Microarrays"

Joel H Rothman, Gina Broitman-Maduro, Morris F Maduro

[West Coast Worm Meeting, 2002]

At the 8-cell stage of C. elegans embryogenesis, the MS and E blastomeres are born. E will give rise to the entire intestine, while MS makes many mesodermal cell types, including body wall muscle and the posterior half of the pharynx. The fates of MS and E are specified by the MED-1,2 GATA transcription factors. While many of the regulators that function in E lineage development have been identified, there is comparatively little known about how MS development proceeds downstream of the MEDs. As a first approximation toward identifying MED target genes in MS, and identifying regulators that might function in the elaboration of the MS lineage, we are using microarray analysis.