Mullan, T.W. et al. (2019) International Worm Meeting "Terminal neuronal cell size is progressively regulated by key neuronal cell fate regulators during embryonic development."

Mullan, T.W., Felton, T., Poole, R.J., Memar, N., Tam, J., Schnabel, R., Yeung, J.T.

[International Worm Meeting, 2019]

Asymmetric cell divisions are a key mechanism by which daughter cells can acquire different fates. In neuronal lineages such divisions often generate unequal daughters. Whether this unequal size directly affects cell fate regulation or is an additional component of the progressive acquisition of final fate is unclear. The latter may be particularly important in C. elegans embryos which lack cell growth. Therefore, control of terminal cell size can likely only be achieved through regulation of daughter size at successive cleavages. To interrogate the mechanisms of neurogenesis we use the embryonic C-lineage which generates two neurons, PVR and DVC, asymmetrically from the anterior Ca branch. Using 4D-lineaging and cell volume measurements we find two successive, dramatically unequal cleavages in the lineage, those of Caa and Caap. These immediately precede expression of the proneural bHLH transcription factor hlh-14/ASCL1, a necessary factor for the generation of PVR and DVC. To address whether daughter size affects neuronal fate acquisition we sort to equalise these cleavages. Employing mutants of a known regulator of unequal cleavages in neuronal lineages, pig-1/MELK, we find equalisation has little impact hlh-14/ASCL1 expression. This both reveals an undescribed role for pig-1/MELK in the C-lineage and indicates that daughter cell size is not a direct input to neuronal fate decisions in the lineage. Assessing what other links may exist between unequal cleavages and fate we analysed daughter size in mutants of key molecular regulators of C-lineage neurogenesis. Interestingly we find the proneural factor hlh-14/ASCL1 controls the second unequal cleavage, with the first regulated by the mediator complex kinase module subunit let-19/MDT-13, which we have recently identified through lineage-based forward genetic screens. Taken together this suggests that successive unequal cleavages, leading to the acquisition of small neuronal cell size in this lineage, are transcriptionally regulated by the same factors that regulate the acquisition of terminal gene expression profiles.

Mullan TW et al. (2024) Development "Control of successive unequal cell divisions by neural cell fate regulators determines embryonic neuroblast cell size."

Kasem O, Poole RJ, Schnabel R, Yeung JT, Tam J, Mullan TW, Memar N, Felton T

[Development, 2024]

Asymmetric cell divisions often generate daughter cells of unequal size in addition to different fates. In some contexts, daughter cell size asymmetry is thought to be a key input to specific binary cell fate decisions. An alternative possibility is that unequal division is a mechanism by which a variety of cells of different sizes are generated during embryonic development. We show here that two unequal cell divisions precede neuroblast formation in the C lineage of C. elegans. The equalisation of these divisions in a pig-1/MELK mutant background has little effect on neuroblast specification. Instead, we reveal let-19/MDT13 as a novel regulator of the proneural bHLH transcription factor hlh-14/ASCL1 and find that both are required to concomitantly regulate the acquisition of neuroblast identity and neuroblast cell size. Thus, embryonic neuroblast cell size in this lineage is progressively regulated in parallel with identity by key neural cell fate regulators. We propose that key cell fate determinants have a novel function to regulate unequal cleavage and therefore cell size of the progenitor cells whose daughter cell fates they then go on to specify.

Mullan, T.W. et al. (2017) International Worm Meeting "4D lineage based screen of temperature sensitive embryonic lethal mutants to identify regulators of left-right asymmetric neurogenesis."

Wademan, R.F., Poole, R.J., Schnabel, R., Mullan, T.W., Felton, T., Kasem, O.

[International Worm Meeting, 2017]

We are interested in uncovering cellular and molecular regulators of proneural gene expression in the context of left-right asymmetric neurogenesis. To achieve this, we focus on the C lineage. Asymmetric expression of the proneural bHLH transcription factor hlh-14/Ascl1 on the left of the lineage is required to produce two glutamatergic tail neurons, DVC and PVR, on the left only. Taking advantage of the invariant cell lineage and single-cell resolution of C. elegans we are performing a 4D-lineage based screen of a collection of over 2000 temperature sensitive embryonic lethal mutants from our collaborator Ralf Schnabel. In wildtype, the cell cycle of the DVC neuroblast(Caapa) is twice the duration of those of its hypoblast cousins. In hlh-14 mutants, neurogenesis is lost with the transformed DVC neuroblast dividing precociously in line with with the hypoblasts, its daughters adopting a hypodermal fate. Through preliminary screening for this precocious division in 150 4D-recordings, comprising 75 mutant strains and secondary screening of our own we have identified three strains of interest. These are and-7(t3224), and-8(t3294) and, and-4(t3200); which has been mapped and cloned and is an allele of let-19, a Mediator complex component. and-4 also displays a symmetrisation of an asymmetric division in the C lineage correlating with loss of neurogenesis (see poster by Terry Felton). Closer study of and-7(t3224) reveals additional ectopic DVC neurons rather than loss. 4D-lineage characterization assessing number and timing of divisions has shown the strain displays highly variable lineage transformations, with the P4 blastomere displaying the greatest and most obvious aberrations. Whole genome sequencing has identified two promising candidates: an endosomal budding gene, pad-1/Dop1p and zinc finger TF sma-9/Schnurri. Loss of pad-1 in embryos is lethal and causes misplacement of cells by an unknown mechanism. This could be due to aberrant lineal origins. In the postembryonic M lineage sma-9 has been shown to antagonize anterior-posterior lineage identity though modulation of TGF beta signaling, yet to our knowledge, a role for TGF beta in the embryo has not been described. Combining modern and classical methods the screen is proving successful in uncovering mutants of the class it was designed for in addition to others. Furthermore, through the identification of let-19 the screen has uncovered another avenue of inquiry in asymmetric control of daughter size in the lineage, a potential cellular regulator of asymmetric neurogenesis in the lineage.

(2024) Development "The people behind the papers - Thomas Mullan and Richard Poole."

[Development, 2024]

Asymmetric cell divisions can produce daughter cells of different sizes, but it is unclear whether unequal cell cleavage is important for cell fate decisions. A new paper in Development explores the role of unequal cleavages in Caenorhabditis elegans embryos. To learn more about the story behind the paper, we caught up with first author Thomas Mullan and corresponding author Richard Poole, Associate Professor of Developmental Biology at University College London, UK.

Felton, T.J. et al. (2017) International Worm Meeting "Cellular and molecular mechanisms of left-right asymmetric neurogenesis."

Schnabel, R., Aldabergenova, A., Memar, N., Yeung, J., Mullan, T.W., Felton, T.J., Kasem, O., Tam, J., Poole, R.J.

[International Worm Meeting, 2017]

We use the C-lineage as a model to study how neuronal potential is asymmetrically inherited to establish left-right (L/R) asymmetric neurogenesis. The C blastomere undergoes 6 successive rounds of cell division to produce L/R symmetric pairs of body wall muscles and hypodermal cells from both sides of the lineage, and two glutamatergic neurons, DVC and PVR, which arise asymmetrically from the left side of the lineage only. Previously, we showed that asymmetric expression of the bHLH proneural gene hlh-14/Ascl1 is required for this L/R asymmetric neurogenesis event. To identify genes that regulate hlh-14 expression and thus affect neuronal specification within the lineage, we have conducted two genetic screens: a forward genetic screen for mutants that fail to express fate makers for DVC and PVR and a 4D-lineage based screen of a collection of temperature-sensitive embryonic lethal mutants. We have isolated eight asymmetric neurogenesis defective (and) mutants. Through mapping-by-sequencing, genetic complementation and rescue experiments, we show that and-6 is an allele of hlh-2, a bHLH transcription factor that heterodimerizes with other bHLH transcription factors, including hlh-14 and that and-4 is an allele of let-19, a member of the Mediator complex. 4D-lineage analysis of the C-lineage reveals that hlh-2 and let-19 mutants exhibit concomitant precocious division of the DVC neuroblast, loss of neuronal cell fate markers, loss of hlh-14 expression and ectopic expression of a hypodermal marker. All of which are phenotypes shared with hlh-14 mutants. We find that let-19 acts during the division of Ca and Caa, and that hlh-2 expression coincides with, and is dependent on, the activity of let-19. These results demonstrate that the Mediator regulates the expression of hlh-2 and hlh-14 during asymmetric neurogenesis. We observe three successive unequal (in size) cell divisions leading up to the production of the DVC neuroblast. We find that let-19 regulates the second of these divisions and in let-19 mutants this division becomes symmetric with a concomitant loss of neuronal potential. The third unequal division is regulated by hlh-14 but not hlh-2 and again there is a concomitant loss of neuronal potential. These results suggest that asymmetric segregation of an unidentified neuronal determinant has a critical role in this lineage and that the Mediator, as well as proneural genes, regulate cell fate and cell size concomitantly. We are currently determining through manipulations of blastomere size if the unequal divisions are an input or output of fate.

Xu, Yan et al. (2013) International Worm Meeting "SYD-1 mediates ventral axon guidance in the HSN neuron of C. elegans."

Xu, Yan, Quinn, Christopher

[International Worm Meeting, 2013]

During development axons navigate towards their targets, giving rise to the neural circuitry that controls behavior. During this navigation process, axons respond to extracellular cues that activate receptors to steer the axon along its trajectory. Guidance receptors are thought to be modular, forming in various combinations that have distinct functions. For instance, the UNC-40 receptor can function alone as a receptor for the UNC-6 guidance cue. Alternatively, UNC-40 and SAX-3 can function together, independently of UNC-6, as a receptor complex for the SLT-1 guidance cue (UNC-40/SAX-3 signaling) [1]. Since these different configurations of UNC-40 have distinct functions, they must have distinct signaling pathways. However, little is known about the pathway that mediates UNC-40/SAX-3 signaling. We have found that UNC-6 independent UNC-40 signaling can be mediated by SYD-1, an intracellular protein that contains a rhoGAP-like domain. We propose that SYD-1 may function with UNC-40 and SAX-3 to mediate UNC-40/SAX-3 signaling to promote the response to the SLT-1 guidance cue. We are currently characterizing the role of SYD-1 in guidance signaling and seeking to determine how it might be regulated to modulate UNC-40/SAX-3 signaling. [1] T.W. Yu et al., Nat. Neurosci. 5:1147-1154 (2002).

Andreia Teixeira-Castro et al. (2005) International Worm Meeting "Using Caenorhabditis elegans as a model for learning the biochemical basis of axon guidance."

Patricia Maciel, Perptua Pinto-do-, Joo Carlos Sousa, Manuel Joo Costa, Andreia Teixeira-Castro

[International Worm Meeting, 2005]

Medical students are often fascinated with the nervous system and with organismal impacts of its dynamics and destruction. The study of molecular cues underlying phenomena such as axonal migration, if properly explored, could catch the students motivation and commitment to learn biochemistry. C. elegans is a suitable, low cost, laboratory model to let moderately skilled students explore the consequences of molecular events at the whole organism level. This work reports on the use of C. elegans to introduce 2nd year medical students to the deleterious effects of gene Knockouts upon response of neurons to extracellular biochemical cues. The main goals of this learning activity were for the students to (1) understand at a basic level the mechanisms of axon guidance and (2) propose possible applications for the usage of knowledge on axon guidance molecules in the treatment of medical pathologies. We divided the activity in three moments. First, students performed behavioral assays to detect movement alterations in C. elegans mutants lacking specific axon guidance molecules. At this moment, students were not aware which genes were mutated. Microscopy observation of abnormal axonal migration patterns of dorsal and ventral cord neurons was also performed. For optimal visualization of neurons, these mutants had been crossed with a pan-neuronal GFP expression strain (Unc-119::GFP). Identification of the specific gene mutated in each strain was then provided to students: Unc-6; Unc-5 and Unc-40. The students were asked to relate abnormal patterns in axon migration/nerve formation with the absent molecular components and the phenotype of mutant strains. Analysis and discussion of a review article about extracellular guidance cues allowed students to further acquire background and consolidate knowledge on this topic (Dynamic regulation of axon guidance. Yu T.W. and Bargmann C.I.; 2001; Nature Neuroscience 4:1169-1176). Finally, students proposed some clinical applications of the recent knowledge concerning the molecular guidance cues and their role in neuronal migration.

Natsuko Watari-Goshima et al. (2004) West Coast Worm Meeting "The C.elegans kinesin-like VAB-8 protein acts with UNC-40/DCC and SAX-3/ROBO to guide axons posteriorly"

Naomi Levy-Strumpf, Joseph G Culotti, Natsuko Watari-Goshima, Gian Garriga

[West Coast Worm Meeting, 2004]

The migrations of neuronal cell bodies and growth cones contribute to the final form and connectivity of the mature metazoan nervous system. While molecules that guide cell and growth cone migrations along the C.elegans D/V axis have been identified, how A/P guidance is regulated remains unresolved. The gene vab-8 is both necessary and sufficient for posteriorly directed migrations, but is not required for anterior or D/V migrations. vab-8 encodes at least two proteins and the larger product, VAB-8L, contains a kinesin-like motor domain at its N-terminus and a coiled-coil domain near its C-terminus. vab-8 is known to function cell autonomously, but how it regulates direction-specific migrations remains unclear. The ALMs are bilaterally symmetric mechanosensory neurons that extend a single axon anteriorly. When VAB-8L was expressed in the ALMs, they often extended their axons posteriorly. This axon rerouting was suppressed by mutations in genes encoding the guidance cue SLT-1, its receptor SAX-3, or the UNC-6 receptor UNC-40. Overexpression of UNC-40 or SAX-3 in the ALM also caused its axon to extend posteriorly, but a vab-8 null mutation suppressed neither UNC-40- nor SAX-3-dependent axon rerouting. Because UNC-40 and SAX-3 have been proposed to act as a heteromeric receptor for SLT-1 (Yu et al., 2002), we asked whether the UNC-40 and SAX-3 receptors act in the same or parallel pathways for VAB-8L-dependent ALM rerouting. A putative null mutation of slt-1 did not enhance the suppression of VAB-8L-dependent axon rerouting by a sax-3 mutation, indicating that SLT-1 acts as a cue for SAX-3 in this process. However, the slt-1 mutation did not suppress SAX-3-dependent axon rerouting, suggesting that SAX-3 requires other ligands to redirect the ALM axon posteriorly. On the other hand, the slt-1 mutation enhanced the suppression of VAB-8L-dependent axon rerouting by an unc-40 mutation. This observation indicates that SLT-1 acts in parallel to UNC-40. Furthermore, a mutation in the gene unc-6 had no effect on the posteriorly directed ALM rerouting caused by overexpression of UNC-40, indicating that UNC-6 does not play a role in this process. Taken together, our observations suggest that VAB-8 directs posterior migrations by regulating the activities of guidance receptor pathways. Before the ALMs extend their axons, their cell bodies migrate posteriorly, and this migration requires VAB-8L. Using the mechanosensory neuron marker mec-4::gfp to sensitize the background and mark the ALMs, we found that both sax-3 and unc-40 mutants showed ALM cell migration defects. We will test whether these receptors act in the same or parallel pathways, and whether they also function with VAB-8 in ALM cell migration. Yu, T.W. et al. (2002) Nature Neuroscience vol.5, no.11, p1147-1154

Bucher EA et al. (1990) Worm Breeder's Gazette "GENETIC ANALYSIS OF ZYGOTICALLY ACTING GENES ESSENTIAL FOR EARLY DEVELOPMENT IN C. ELEGANS"

Bucher EA, Greenwald IS

[Worm Breeder's Gazette, 1990]

We are using a genetic mosaic screen for mutations in zygotically acting genes that are essential for early development. This strategy allows us to distinguish gene products whose functions are required in specific lineages from gene products required for general cell viability (housekeeping functions). In contrast to other approaches, mosaic analysis does not rely upon lethal phenotypes, but upon focus of gene activity, and therefore may allow us to identify mutations in genes whose lethal phenotypes we may not anticipate. We hypothesized that this approach is reasonable since we can make the following predictions about the properties that different classes of essential, zygotically active genes may exhibit: Class I. genes whose functions are required in all lineages (housekeeping); Class II. genes whose functions are required everywhere but expression in part of the lineage is sufficient (diffusible housekeeping); Class III. genes whose functions are involved in determination and differentiation and required only in specific lineages; Class IV. genes having essential functions in certain lineages and nonessential functions in others. We have generated 31 independent lethal mutations in the strain ncl- 1 glp-1; d by Judith Austin). The EMS induced lethal mutations are linked to these recessive markers and complemented by the free duplication qDp3. The visible markers unc-36 and glp-1 have specific foci of activity in ABp and P4, respectively, allowing a rapid visual screen for rare somatic losses of qDp3 during early cell divisions. Losses of qDp3 are determined precisely using the cell autonomous nucleolar marker ncl-1 ( E. Hedgecock) visualized under Nomarski optics. The pattern of rare viable mosaics should reflect the requirement for gene activity. Mosaic analysis among a set of the 31 lethal strains that we have generated has distinguished mutations consistent with the predicted classes: some lethal strains never generate mosaics (Class I); a few strains generate viable mosaics in a restricted part of the lineage ( Class III); and two strains generate viable mosaics in a restricted part of the lineage and show an additional specific phenotype (Class IV); (see below). Although we have not yet seen any mosaic patterns consistent with Class II gene products, Bob Herman (personal communication) and Wightman and Ruvkun (WBG (1989) 11: 48) have observed mosaic patterns that we consider to be of this class. Strain GS192 appears to contain a mutation in a Class IV gene that has an essential function in the P1 lineage and a nonessential function in the AB lineage. Rare viable mosaics (picked as Uncs or semi Uncs) in GS192 result from losses in AB, ABp, ABpl, and ABpr . In contrast, we have never isolated a viable mosaic resulting from loss in the P1 lineage, suggesting that the expression of this gene product is absolutely required in these cells. However, a nonessential function was revealed by the abnormal morphology of neurons derived from AB in mosaic animals lacking qDp3 in these AB derived cells. Neurons derived from AB do not exhibit their characteristic granular appearance and do not have visible Nc1 nucleoli. GS161 also appears to encode a Class IV product. Unc and semi-Unc mosaic animals result from losses in either ABp, ABpl, or ABpr. This pattern suggests that expression of the gene product is absolutely required in the P1 lineage as well as in the ABa lineage. A nonessential function was revealed by abnormal vulval formation in mosaic animals. This observation is especially interesting since cells giving rise to the vulva descend from ABp. All mosaics are Egl-; some mosaics have no vulva, whereas other mosaics have an abnormal vulva. Preliminary mapping data place this allele left of ncl-1 where, to our knowledge, no genes affecting vulval development have been previously identified. The variable vulval phenotype may indicate either that this is not a null allele or, alternatively, may reflect some aspect of this gene product's requirement in the vulval lineages. These data argue that this genetic mosaic screen may indeed allow us to identify lethal mutations in zygotically expressed genes that are required for basic developmental processes. Furthermore, mosaic analysis not only allows us to distinguish among genes products having different foci of activity, but may also allow us to identify mutations in genes which may be too pleiotropic to recognize by criteria such as terminal phenotype. Some gene products of interest to us may have multiple, but specific roles in development (Cline, T.W. (1989) Cell 59: 231-234.) and thus may be classified as pleiotropic by classical criteria but may be identified in this genetic mosaic strategy as interesting. (Note: we have detected recombination between qDp3 and the chromosomes with some growth advantage for recombinants.)