Bob Goldstein (2006) Development & Evolution Meeting "Cell Fate Determination in C. elegans"

Bob Goldstein

[Development & Evolution Meeting, 2006]

The C. elegans embryo has been a remarkably useful model for studying diverse topics in cell and developmental biology. A key advantage to scientists who use C. elegans as a model system is the potential to readily combine a large number of useful techniques, including techniques of modern microscopy, forward and reverse genetics, molecular biology and biochemistry. In this talk, I will review just a few of the many C. elegans experiments that have made significant impacts in understanding embryonic cell fate determination mechanisms, emphasizing experiments that have creatively combined unusual techniques with established techniques. I will also present some of my own lab's recent experiments using C. elegans embryos to understand one key aspect of cell fate determination -- how cells become polarized.

Bob Goldstein (2003) International Worm Meeting "Polarization of a single cell by asymmetric Wnt signaling in the presence of Src signaling"

Bob Goldstein

[International Worm Meeting, 2003]

Cell interactions can change the fates of large groups of cells during development. In recent years, it has become clear that developmental cell interactions can also polarize single cells. Such polarization events often depend on Wnt signals, which are known to be important players in both development and cancer cell biology. It has not been clear whether Wnt signals truly act as asymmetric cues to polarize cells, or alternatively whether Wnt signals simply need to be present while other signals polarize cells. For example, expressing Wnt signals in worms either ubiquitously or in an abnormal position can rescue normal Wnt-dependent cell polarity (Herman et al 1995, Whangbo et al 2000) -- suggesting that the normal position of the Wnt signal may be unimportant in some cases of cell polarization. In no organism has it been shown that the asymmetric position of a Wnt signal is essential for polarizing a cell. This can be tested by placing signals at specific positions on a responding cell, using cell manipulations. Work from several labs has shown that at the four-cell stage in C. elegans, a Wnt signal and a Src signal from the P2 cell act together to polarize the EMS cell. These signals position gut identity in one side of EMS, and they also align the mitotic spindle in EMS. To test whether one signal acts as an asymmetric cue to polarize EMS, and whether the other signal might act as a permissive signal in EMS cell polarization, experiments were set up in which the Wnt and Src signals were presented to different positions on EMS. Mutant P2 cells were used as sources of Wnt and Src signals, and a GFP:histone transgene was used to confirm mutant cell identities. Gut induction was assayed after Wnt and Src signals were presented to opposite sides of uninduced EMS cells: gut was induced only on the side presented with the Wnt signal. Spindle orientation was assayed after Wnt and Src signals were presented at orthogonal positions on uninduced EMS cells: the spindle consistently oriented in line with the Wnt signal. Other experiments confirmed a previous report that the Src signal is essential for spindle orientation but is a redundant signal for gut induction (Bei et al 2002). These results indicate that a Wnt signal can act as an asymmetric cue to polarize a cell, that a Src signal can act as an essential signal without providing positional information, and that Wnt and Src signals are capable of working together from different positions.

Hibshman, Jon et al. (2021) International Worm Meeting "LEA motifs promote desiccation tolerance in vivo"

Hibshman, Jon, Goldstein, Bob

[International Worm Meeting, 2021]

Cells and organisms typically cannot survive in the absence of water. However, there are some notable exceptions, including animals such as nematodes, tardigrades, rotifers, and some arthropods. One class of proteins known to play a role in desiccation resistance is the late embryogenesis abundant (LEA) proteins. These largely disordered proteins protect plants and animals from desiccation. A multitude of studies have characterized stress-protective capabilities of LEA proteins in vitro and in heterologous systems. However, the extent to which LEA proteins exhibit such functions in vivo, in their native contexts in animals, is unclear. Furthermore, little is known about the distribution of LEA proteins in multicellular organisms or tissue-specific requirements in conferring stress protection. To study the endogenous function of an LEA protein in an animal, we created a true null mutant of C. elegans LEA-1, as well as endogenous fluorescent reporters of the protein. We confirmed that C. elegans lacking LEA-1 are sensitive to desiccation. LEA-1 mutant animals were also sensitive to heat and osmotic stress and were prone to protein aggregation. During desiccation, LEA-1 expression increased and became more widespread throughout the body. LEA-1 was required at high levels in body wall muscle for animals to survive desiccation and osmotic stress. We identified minimal motifs within C. elegans LEA-1 that are sufficient to increase desiccation survival of E. coli. To test whether such motifs are central to LEA-1's in vivo functions, we then replaced the sequence of lea-1 with these minimal motifs and found that C. elegans survived mild desiccation and osmotic stress at the same levels as worms with the full-length protein. Our results provide insights into the endogenous functions and expression dynamics of an LEA protein in a multicellular animal. The results show that LEA-1 buffers animals from a broad range of stresses. Our identification of LEA motifs that can function in both bacteria and in a multicellular organism suggests the possibility of engineering LEA-1-derived peptides for optimized desiccation protection.

Dickinson, Daniel J. et al. (2015) International Worm Meeting "Single molecule analysis of PAR protein complexes during cell polarization."

Goldstein, Bob, Dickinson, Daniel J.

[International Worm Meeting, 2015]

The conserved PAR protein system mediates polarization in a wide variety of animal cell types including epithelia, neurons and stem cells. The first division of the C. elegans zygote is a valuable model system for studying how PAR proteins mediate cell polarization. This cell division is asymmetric, with the anterior and posterior daughter cells adopting different fates. PAR-3, PAR-6, atypical Protein Kinase C (aPKC) and CDC-42 localize to the anterior of the zygote prior to division, while PAR-1, PAR-2 and Lethal Giant Larvae (LGL-1) localize to the posterior. All of these proteins are required (some redundantly) for a normal polarized division. Although the localization and genetic requirements of PAR proteins have been studied in detail, a fundamental unanswered question is how these proteins are organized biochemically into a signaling network that can mediate cell polarization. Although some pairwise protein-protein interactions between members of the PAR system have been demonstrated in vitro, it is how these interactions are spatially and temporally regulated in vivo to generate cell polarity. To address this, we have developed a method that allows us to interrogate protein-protein interactions in individual, precisely staged C. elegans embryos. Single embryos are crushed in microfluidic chambers, generating lysates in a small volume with minimal dilution. Protein complexes are captured inside this device by antibodies, and can then be counted using single-molecule TIRF microscopy. We are using this assay to test which of the reported interactions between members of the PAR system occur in vivo, and whether these interactions are temporally regulated during the establishment and maintenance of cell polarity. .

Dickinson, Daniel J. et al. (2015) International Worm Meeting "Rational design of protein coding sequences that evade piRNA-mediated germline silencing."

Dickinson, Daniel J., Goldstein, Bob

[International Worm Meeting, 2015]

In the C. elegans germline, multiple mechanisms act to recognize and silence foreign DNA. Because these pathways can operate on experimentally introduced transgenes, they pose a technical challenge to cell biologists studying the germline and early embryo. Developing strategies to overcome or bypass silencing of transgenes would facilitate research in multiple fields that use C. elegans embryos as a model system. One pathway that operates to silence single-copy foreign sequences involves recognition of the foreign DNA by genomically encoded 21U-RNAs, which initiate silencing through the Piwi Argonaute PRG-1 (Shirayama et al. 2012). The diversity of 21U-RNAs is high enough to target essentially any DNA sequence. Endogenous germline genes are protected from silencing by a second pathway that involves sequence-dependent licensing by 22G-RNAs bound to the Argonaute CSR-1 (Seth et al. 2013). Newly generated transgenes can adopt either an expressed or a silent state as a result of competition between these two pathways. In some cases, recognition of the GFP portion of a transgene by the PRG-1 pathway can lead to complete silencing. Because silencing is sequence-dependent, we thought it should be possible to design protein coding sequences that evade recognition by PRG-1 and thereby escape silencing. Designing sequences that lack 21U-RNA recognition sites is difficult because the number of 21U-RNAs is very large, and their base pairing specificity (i.e., mismatch tolerance) has not been well characterized. However, we reasoned that bona fide germline-expressed genes should be depleted for high-affinity 21U-RNA binding sites, and might also be enriched for 22G-RNA binding sites that could facilitate expression. Therefore, we developed an algorithm that constructs a coding sequence for a given protein of interest by assembling short "words" found in germline-expressed genes. We refer to coding sequences generated in this way as "germline optimized." We constructed synthetic transgenes to test whether germline-optimized sequences are resistant to silencing. We obtained robust germline expression of bacterial proteins that were otherwise efficiently silenced. Moreover, by using a germline-optimized GFP coding sequence, we were able to obtain expression of gfp::cdk-1, a model transgene that is particularly prone to silencing. We have produced a web-based tool that allows users to evaluate the degree of germline optimality of existing transgenes and to design germline-optimized coding sequences for any protein of interest. .

Sawa, Hitoshi et al. (2019) International Worm Meeting "Reciprocal control of Wnt and Frizzled asymmetry during cell polarization."

Goldstein, Bob, Sawa, Hitoshi, Onami, Mayumi, Pani, Ariel, Matsuo, Misato

[International Worm Meeting, 2019]

Wnts are secreted signaling proteins that regulate cell polarity in many contexts. Wnt signaling between neighboring cells can instructively regulate cell polarity, but how a long-range Wnt signal can regulate polarity at a distance is unclear. To address how cells interpret Wnt gradient information to generate cell polarity, we turned to C. elegans seam cells, which polarize and asymmetrically divide in response to three Wnts: egl-20, cwn-1, and cwn-2. These Wnts are partially redundant in most seam cells despite different expression patterns, and mis-expression experiments suggested that these Wnts may be permissive signals for seam cell polarization (Yamamoto, et. al., 2011). Here, we report that Wnts also have instructive functions in seam cell polarization and that these roles may depend on an unexpected positive feedback reinforcement of asymmetric Wnt and Frizzled localizations to the posterior ends of polarizing cells. We first found that Wnts can instructively regulate seam cell polarity in the absence of the Frizzled homolog LIN-17: Wnt mis-expression in a lin-17 mutant enhances seam cell polarity reversals. This result suggests a permissive function for LIN-17 might obscure an underlying instructive role for Wnt. Based on the finding that EGL-20/Wnt localizes to the posterior ends of many seam cells (Pani and Goldstein, 2018), we hypothesized that asymmetric Wnt enrichment could explain how a long-range Wnt gradient might instruct cell polarity. To investigate the cell biological basis for instructive Wnt signaling, we quantified localization of endogenously tagged Wnts in seam cells prior to their first asymmetric division. We found that EGL-20 and CWN-1 are enriched at the posterior ends of most seam cells, including cells far from the posterior Wnt sources. We discovered that EGL-20/Wnt and MOM-5/Frizzled are reciprocally required for posterior ligand and receptor enrichment in seam cells. We propose that positive feedback can reinforce subcellular asymmetries in Wnt and Frizzled localizations, which allows cells to robustly interpret Wnt gradient information to instruct cell polarity and fates following asymmetric divisions.

Shemer, Gidi et al. (2009) International Worm Meeting "Driving with a clutch: cadherin acts with Rac signaling to regulate cell movements during gastrulation."

Goldstein, Bob, Shemer, Gidi, Roh, Minna, McClellan, Joe

[International Worm Meeting, 2009]

We have studied early gastrulation in C. elegans as a model to understand how adhesion proteins interact with intracellular processes to regulate cell movements. C. elegans gastrulation starts at the 26-cell stage, with the internalization of the two endodermal precursors (E cells), accompanied by movements of the neighboring cells to cover the exposed ventral space. This process requires that the E cells adopt a proper cell fate and develop apico-basal polarity. Apical accumulation of activated non-muscle myosin in the E cells leads to apical constriction, resulting in their inward movements. We have taken a reverse genetic approach by knocking down candidate adhesion proteins, as well as members of signaling pathways that may interact with the adhesion machinery and the cytoskeleton. We have found that knockdown of HMR-1/cadherin together with the Rac activator CED-5 leads to failure of the E cells to internalize. Moreover, knockdown of other components of the cadherin-catenin complex, HMP-1 and HMP-2, in a ced-5(-) background resulted in similar gastrulation defects. We also found that CED-12 and CED-2, proteins that act with CED-5 as part of a RacGEF complex later in development, also function in gastrulation. Finally, we found that CED-10/Rac shows similar gastrulation defects. Thus, cadherin-catenin proteins act together with Rac signaling to allow gastrulation movements. Cell fate, polarity, and apical accumulation of myosin in the E cells are not compromised in hmr-1;ced-5 mutant embryos. To better address the dynamics of apical constriction, we imaged wild type and mutant embryos for movement of myosin foci with respect to the zones where E cells contact their neighboring cells. Surprisingly, we have revealed that in wild type embryos, myosin undergoes centripetal movements along the apical cortex even before apical surfaces begin to shrink. This first phase of myosin movement is then followed by a second phase, where the contact zones between cells begin to narrow in concert with the myosin movements until apical constriction is completed and cells internalize. In contrast to wild type embryos, hmr-1;ced-5 mutants showed defects in membrane dynamics. While centripetal movements of myosin took place normally, these movements were not followed by narrowing of the contact zones in the second phase, resulting in failure of the E cells to internalize. Our results suggest that gastrulation in C. elegans involves a clutch-like mechanism, whereby myosin motors move for some time without causing apical constriction. Only after this period do the cadherin-catenin complex and Rac signaling act together to serve as force carriers of cortical contraction to internalize the gastrulating cells.

Osborne Nishimura, Erin et al. (2013) International Worm Meeting "Single-blastomere transcriptome profiling after the first embryonic division."

Osborne Nishimura, Erin, Lieb, Jason D., Goldstein, Bob, Werts, Adam, Zhang, Jay C.

[International Worm Meeting, 2013]

We are interested in understanding how reproducible patterns of RNA inheritance and abundance arise in dividing cells, and for what purpose. The C. elegans early embryo represents a unique biological system to study this problem because the lack of zygotic transcription removes the potential complication of transcriptional fluctuations. Following fertilization and prior to the onset of zygotic transcription, the C. elegans zygote cleaves asymmetrically to create the anterior AB and posterior P1 blastomeres. To identify asymmetrically distributed transcripts, we dissected the AB and P1 blastomeres and performed low-input RNA-seq. Our results are consistent with a recently published study with a similar experimental design (Hashimshony et al, 2012 Cell Reports). We developed a scoring method for ranking transcripts, verified asymmetric patterns using qRT-PCR and in situ hybridization, and narrowed our analysis to a subset of transcripts that represent the most asymmetric RNAs in each cell at the 2-cell stage. AB and P1 are the founders of two lineages with distinct fates. We found that transcripts enriched in AB tend to encode proteins that function in mitosis and epithelium development, whereas transcripts segregated to the P1 are associated with translational regulation. To test whether any of the genes in our AB-enriched or P1-enriched subsets play a functional role in asymmetric cell division or downstream embryonic patterning, we performed controlled RNAi phenotyping and video microscopy to categorize embryological defects. Using these methods, we identified F32D1.6, an AB-enriched (anterior) transcript with a role in anterior-specific morphological events. In addition, two posterior enriched transcripts, T04A8.7 and puf-3, were found to exhibit noteworthy early embryological defects. We are currently identifying sequence elements and RNA binding proteins that are required for this process with the goal of elucidating the mechanisms behind asymmetric RNA partitioning.

Siddiqui SS et al. (1997) International C. elegans Meeting "ARMADILLO BY A DOZEN: 12 ARMADILLO REPEATS IN CeKAP-1, A KINESIN ASSOCIATED PROTEIN"

Ali Khan L, Siddiqui ZK, Siddiqui SS

[International C. elegans Meeting, 1997]

Previously we have reported cloning of the osm-3 gene, encoding a kinesin like protein (Shakir et al., 1993). OSM-3 shows high homology to the sea urchin spKRP85 and spKRP95 kinesin like proteins (Kinesin II). Wademan et al. (1996) have recently reported a non-motor kinesin associated protein KAP115 from sea urchin which co-purifies with kinesinII holoenzyme. We have isolated a cDNA clone (called CeKAP-1) from a nematode cDNA library, corresponding to the nematode homologue of the spKAP115 protein, that maps on the cosmid F08F8.3 (Wademan et al., 1996). Similar multi-meric complexes have been reported in mouse KIF3A-KIF3B-KAP3 (Hirokawa, 1996), and in Drosophila (KLP64D and KLP68D) (Goldstein, 1993). Gindhart and Goldstein (1997) noted that these KAP proteins harbor several repeats of the Drosophila armadillo motif. Indeed, the nematode CeKAP-1 when analyzed shows twelve contiguous armadillo repeats in the KAP-1 primary sequence, unlike spKAP115 protein that contains a total of 10 armadillo repeats, one repeat is separtaed from the other nine, which are tandem repeats. Since armadillo like repeats have been found in a variety of proteins which mediate intracellular signalling and protein interactions, a role for CeKAP-1 protein in regulating the cargo binding of the OSM-3/KLP-11( Kinesin II ) motor proteins, is worth examining. We are looking for mutants affected in the CeKAP-1 and KLP-11 genes which may show interactions with the osm-3 mutants.

Osborne Nishimura, Erin et al. (2015) International Worm Meeting "Cell-specific and sub-cellular mRNA patterning in early embryogenesis."

Lieb, Jason D., Wertz, Adam D., Goldstein, Bob, Tintori, Sophie, Zhang, Jay C., Osborne Nishimura, Erin

[International Worm Meeting, 2015]

We are interested in understanding how the transcriptomes of embryonic cells are regulated at single-cell and sub-cellular levels and how key mRNA transcripts promote lineage-specific development. Using RNA-seq on pooled, hand-dissected AB and P1 blastomeres, we identified over 200 transcripts that were asymmetrically patterned after the first cell division in the C. elegans zygote. We confirmed asymmetric abundance patterns for a subset of these transcripts using smFISH. smFISH also revealed heterogeneous subcellular patterning of some transcripts including chs-1 and bpl-1 that were granularly distributed in cells of the P lineage. We screened asymmetric transcripts for embryonic defects using RNAi and identified the gene neg-1 as an AB-enriched (anterior) transcript that was required for proper morphology of anterior tissues. In addition, we identified sequence motifs and features that were over-represented in asymmetric transcripts, giving us clues to regulatory mechanisms. Current research includes efforts to unveil mechanisms responsible for cellular and subcellular patterns of mRNA abundance. Further, we are using single-cell transcriptome profiling to identify blastomere-specific transcripts in the 4-cell to 16-cell stages of embryogenesis.