Mizeracka, Karolina et al. (2017) International Worm Meeting "Using C. elegans to understand glial diversity."

Heiman, Maxwell, Mizeracka, Karolina, Bulyk, Martha, Rogers, Julia, Shaham, Shai

[International Worm Meeting, 2017]

Glial cells comprise half of our nervous system and play critical roles in its function, yet basic understanding of how glia arise during development remains surprisingly limited. For example, what molecules establish glial fate and how do glia become different from each other? To address these questions, we have turned to the well-defined and experimentally tractable nervous system of C. elegans as a simple model of glial cell type diversity. We have focused on specification of two different glial cells - the sheath and socket cells of the amphid sense organ. Although these two glial cells are in close proximity, they are morphologically and molecularly distinct: the sheath is larger, more highly secretory, and expresses different molecular markers than the socket. In a forward genetic screen, we identified sheath and socket fate defects caused by a missense mutation (W201G) in the DNA-binding domain of a conserved Forkhead transcription factor, UNC-130. Among mutant animals, ~25% lose expression of sheath-specific markers and ~35% independently exhibit ectopic expression of socket-specific markers in other cells. In contrast, animals bearing a different missense mutation (R218C) in a nearby region of the DNA-binding domain exhibit sheath fate defects yet do not have "extra" socket glia. These results suggest that UNC-130 affects sheath and socket specification by different means, for example, by binding to distinct classes of DNA sequence motifs. To test this hypothesis, we used protein-binding microarrays to measure the affinity of wild-type, W201G, and R218C variants of UNC-130 for all possible 10-mer DNA sequences. Wild-type UNC-130 preferentially bound motifs consistent with canonical Forkhead sites. UNC-130(W201G) exhibited almost no specific sequence binding, while UNC-130(R218C) retained the ability to bind high-affinity motifs but was severely impaired in its binding to some lower-affinity motifs. These results suggest a model in which UNC-130 binds distinct DNA motifs to control amphid sheath and socket glial fate, with both activities being lost in UNC-130(W201G) and only sheath-specific activities being lost in UNC-130(R218C). Importantly, the vertebrate homolog of UNC-130, FoxD3, has also been implicated in specifying glial fates. We found that expression of human FoxD3 completely rescues the glial fate defects of unc-130 mutants, suggesting that the glial specification pathways we uncover in C. elegans may be well conserved in humans.

Mizeracka, Karolina et al. (2021) International Worm Meeting "Lineage-specific paths to the same cell type"

Murray, John, Shaham, Shai, Bulyk, Martha, Rumley, Jonathan, Heiman, Maxwell, Mizeracka, Karolina, Rogers, Julia

[International Worm Meeting, 2021]

Convergent differentation - in which the same cell type is produced by multiple unrelated developmental lineages - provides a striking exception to the classical framework of cell fate specification, in which a given cell type arises exclusively from a single lineage. Convergent differentiation has long been appreciated in C. elegans and is also prevalent in vertebrate development, most notably in the neural crest. However, the mechanisms that dictate whether lineages will take on divergent or convergent transcriptional paths are not well understood. We focused on the development of six radially symmetric glial cells, called ILso glia, that are produced by distinct lineages. Through an unbiased genetic screen, we found that the Forkhead transcription factor UNC-130/Foxd3 is absolutely required for the specification of the dorsal, but not the ventral or lateral, ILso glia. Through lineaging and timed rescue experiments, we found that UNC-130 acts transiently as a transcriptional repressor in progenitors and newly-born terminal cells of the dorsal ILso lineage. UNC-130 can be functionally replaced with the neural crest determinant Foxd3, suggesting it may play a conserved role in lineages that undergo convergent differentiation. Surprisingly, in addition to loss of the dorsal ILso, we find that unc-130 mutants also have ectopic formation of another glial type, the AMso glia. Ectopic AMso glia take on the morphology and functions of their endogenous counterparts, including dividing in males to produce an extra male-specific MCM neuron. However, they consistently arise in the wrong anatomical position, suggesting they may be produced by mis-specification of cells from the dorsal ILso lineage. We find that the neuronal determinant UNC-86/Brn3 is spuriously upregulated in the dorsal ILso lineage in unc-130 mutants and is required for the ectopic AMso glia to form, suggesting that one role of UNC-130 may be to prevent expression of UNC-86 in this lineage. Together, these data suggest that convergent differentiation involves distinct transcriptional paths leading to the same cell type, and highlight the importance of transiently-acting transcriptional repressors that may constrain progenitor fates.

Mizeracka, Karolina et al. (2013) International Worm Meeting "Developing a cell contact sensor for tracking neuron-glia interactions in vivo."

Mizeracka, Karolina, Heiman, Maxwell

[International Worm Meeting, 2013]

The mechanism by which neurons selectively recognize their glial partners is an important and largely unexplored question in neurobiology. To begin to address this question, we are developing a cell contact sensor designed to report on the interaction of adjacent cells. Inspired by the GRASP system developed by Bargmann and colleagues that can be used to label synaptic contacts by the extracellular reconstitution of a GFP molecule, we aim to develop a contact sensor that will trigger a transcriptional readout in a cell of interest, providing a more permanent mark. In order to translate an external interaction to a change in intracellular gene expression, we have modeled our contact sensor on the well-characterized Delta-Notch ligand-receptor signaling pathway, a naturally occurring system in which cell-cell contact triggers transcriptional activity. In brief, interaction of an artificial ligand-receptor pair on the surfaces of adjacent cells will result in intramembrane proteolysis of the receptor, releasing a cytoplasmic transcriptional effector, and thereby genetically marking the receiver cell. First, we will test our reporter system in S2 Drosophila cells, ensuring that interaction between our artificial ligand and receptor results in cleavage of the receptor and activation of a transcriptional reporter. Next, we will test our reporter system in vivo, focusing initially on the stereotyped and highly selective interactions between the amphid sensory neuron AFD and the amphid sheath glial cell that surrounds the AFD dendrite tip. Specifically, we will express an artificial ligand on the surface of the AFD neuron and an artificial receptor on the surface of the amphid sheath glial cell. Contact between this neuron and its glial cell will result in a transcriptional output, activating a fluorescent reporter in the glial cell. This system will allow us to screen for "miswiring" mutants in which an amphid neuron improperly associates with a non-amphid glial cell, as a means to identify factors that mediate neuron-glia recognition. Once established, this contact sensor could be readily applied to report on any cell-cell interaction, in C. elegans or other animals.

Mizeracka, Karolina et al. (2019) International Worm Meeting "Conserved molecular pathways establish glial diversity in C. elegans."

Rogers, Julia, Shaham, Shai, Mizeracka, Karolina, Bulyk, Martha, Heiman, Maxwell

[International Worm Meeting, 2019]

Glia comprise half of the human nervous system and are essential for its function. Recent transcriptional profiling studies have revealed that these cells are highly heterogeneous with numerous subtypes in different parts of the nervous system. However, the molecular pathways that establish glial cell types remain unknown. Because C. elegans has provided a platform for dissecting the regulation of neuron specification, we reasoned that it would be a powerful system for addressing the novel question of glial specification. We identified a role for the transcription factor, UNC-130/FoxD3, in specifying two distinct glial types through an unbiased genetic screen. In unc-130 mutants, animals lose expression of fate markers specifically in the dorsal inner labial sockets (ILsoD), but not their lateral or ventral counterparts. Surprisingly, the missing ILsoD cells appear to convert to a different glial fate, the amphid socket (AMso). Another glial cell in the ILsoD lineage, the amphid sheath (AMsh), is also affected. Interestingly, we found that missense mutations at different positions in the UNC-130 DNA-binding domain exhibit graded phenotypic severity. To determine how these mutations affect DNA binding, we used protein-binding microarrays representing all 10-mer DNA sequences to define the UNC-130 consensus motif and to measure the binding affinity of mutant UNC-130. We found that levels of UNC-130:DNA binding in vitro correlated with phenotypic severity in vivo, suggesting that some glial types exhibit more stringent requirements for UNC-130 activity. Since Forkhead transcription factors can act either as transcriptional activators or repressors, we used in vitro and in vivo assays to determine that ILsoD and AMsh fates both require UNC-130 repressor activity. This suggests that UNC-130 might repress factors that would otherwise disrupt glial specification. Consistent with this idea, we identified two transcription factors, RNT-1 and UNC-86, that suppress the ectopic AMso and loss of AMsh phenotypes respectively. Importantly, the vertebrate homolog of UNC-130, FoxD3, completely rescues the mutant glial fate defects in C. elegans. Further, the homologs of FoxD3/UNC-130, Runx1/RNT-1, and Brn3/UNC-86 are involved in specification of neuron and glial fates in the vertebrate neural crest lineage, providing evidence that the molecular mechanisms we have identified in C. elegans will shed light on glial specification in vertebrates.

Mizeracka, Karolina et al. (2021) International Worm Meeting "Two of the 30 EGF domains in FBN-1/Fibrillin are required for sensory dendrite extension"

Heiman, Maxwell, Mizeracka, Karolina, Shaham, Shai

[International Worm Meeting, 2021]

Many extracellular matrix (ECM) components are large proteins composed of highly repetitive domains. For example, FBN-1/Fibrillin is a ~2700 aa protein with 30 EGF-like domains. In principle, such repetitive domains could function redundantly, for example to cross-link other matrix components, or they could be distinct modules with specialized functions. In previous studies, disruption of fbn-1 with a large internal deletion (tm290) or RNAi caused almost complete lethality and severe molting defects. In contrast, through a genetic screen for mutants affecting amphid dendrite morphogenesis, we identified two alleles of fbn-1 (ns67 and ns283) that introduce missense mutations within 32 aa in the first two N-terminal EGF-like domains (EGF1-2). These mutants cause weakly penetrant defects in amphid dendrite extension (8% and 19% defective, respectively), with minimal lethality or molting defects, suggesting that the EGF1-2 domains perform a specialized function related to dendrite extension. To further understand this, we generated a small in-frame deletion encompassing EGF1-2 (syb239) and a complete deletion of fbn-1 (syb240) using CRISPR/Cas9. Consistent with our original mutant analysis, syb239 has weakly penetrant amphid dendrite extension defects (8% defective), while syb240 exhibits severe embryonic lethality, supporting the idea of a specialized role for EGF1-2. Amphid dendrite extension also requires another ECM protein, DYF-7, which is produced by the amphid neurons and forms "caps" at the dendrite endings that prevent rupture of the developing neuron-glia sensory epithelium. FBN-1 is expressed by hypodermal cells at this stage, and we find that the EGF1-2 domains are required for proper formation of DYF-7 caps. These results show that distinct domains within FBN-1 may be specialized to support different aspects of its function, and suggest that even highly repetitive domains in ECM proteins may reflect modularity rather than redundancy.