Live imaging of retinotectal mapping reveals topographic map dynamics and a previously undescribed role for Contactin 2 in map sharpening.

Organization of neuronal connections into topographic maps is essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotectal mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum, leading to the sharpening of the retinotopic map along the antero-posterior axis. Finally, using a CRISPR-mediated mutagenesis approach, we demonstrate that the refinement of nasal retinal projections requires the adhesion molecule Contactin 2. Altogether, our study provides the first analysis of a topographic map maturing in real time in a live animal and opens new strategies for dissecting the molecular mechanisms underlying precise topographic mapping in vertebrates.

Counteracting epigenetic mechanisms regulate the structural development of neuronal circuitry in human neurons.

Autism spectrum disorders (ASD) are associated with defects in neuronal connectivity and are highly heritable. Genetic findings suggest that there is an overrepresentation of chromatin regulatory genes among the genes associated with ASD. ASH1 like histone lysine methyltransferase (ASH1L) was identified as a major risk factor for ASD. ASH1L methylates Histone H3 on Lysine 36, which is proposed to result primarily in transcriptional activation. However, how mutations in ASH1L lead to deficits in neuronal connectivity associated with ASD pathogenesis is not known. We report that ASH1L regulates neuronal morphogenesis by counteracting the catalytic activity of Polycomb Repressive complex 2 group (PRC2) in stem cell-derived human neurons. Depletion of ASH1L decreases neurite outgrowth and decreases expression of the gene encoding the neurotrophin receptor TrkB whose signaling pathway is linked to neuronal morphogenesis. The neuronal morphogenesis defect is overcome by inhibition of PRC2 activity, indicating that a balance between the Trithorax group protein ASH1L and PRC2 activity determines neuronal morphology. Thus, our work suggests that ASH1L may epigenetically regulate neuronal morphogenesis by modulating pathways like the BDNF-TrkB signaling pathway. Defects in neuronal morphogenesis could potentially impair the establishment of neuronal connections which could contribute to the neurodevelopmental pathogenesis associated with ASD in patients with ASH1L mutations.

Teneurin trans-axonal signaling prunes topographically missorted axons.

Building precise neural circuits necessitates the elimination of axonal projections that have inaccurately formed during development. Although axonal pruning is a selective process, how it is initiated and controlled in vivo remains unclear. Here, we show that trans-axonal signaling mediated by the cell surface molecules Glypican-3, Teneurin-3, and Latrophilin-3 prunes misrouted retinal axons in the visual system. Retinotopic neuron transplantations revealed that pioneer ventral axons that elongate first along the optic tract instruct the pruning of dorsal axons that missort in that region. Glypican-3 and Teneurin-3 are both selectively expressed by ventral retinal ganglion cells and cooperate for correcting missorted dorsal axons. The adhesion G-protein-coupled receptor Latrophilin-3 signals along dorsal axons to initiate the elimination of topographic sorting errors. Altogether, our findings show an essential function for Glypican-3, Teneurin-3, and Latrophilin-3 in topographic tract organization and demonstrate that axonal pruning can be initiated by signaling among axons themselves.

To Stick or Not to Stick: The Multiple Roles of Cell Adhesion Molecules in Neural Circuit Assembly.

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.

Inappropriate cathepsin K secretion promotes its enzymatic activation driving heart and valve malformation.

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-ß-related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.

Deficiency in the endocytic adaptor proteins PHETA1/2 impairs renal and craniofacial development.

A critical barrier in the treatment of endosomal and lysosomal diseases is the lack of understanding of the in vivo functions of the putative causative genes. We addressed this by investigating a key pair of endocytic adaptor proteins, PH domain-containing endocytic trafficking adaptor 1 and 2 (PHETA1/2; also known as FAM109A/B, Ses1/2, IPIP27A/B), which interact with the protein product of OCRL, the causative gene for Lowe syndrome. Here, we conducted the first study of PHETA1/2 in vivo, utilizing the zebrafish system. We found that impairment of both zebrafish orthologs, pheta1 and pheta2, disrupted endocytosis and ciliogenesis in renal tissues. In addition, pheta1/2 mutant animals exhibited reduced jaw size and delayed chondrocyte differentiation, indicating a role in craniofacial development. Deficiency of pheta1/2 resulted in dysregulation of cathepsin K, which led to an increased abundance of type II collagen in craniofacial cartilages, a marker of immature cartilage extracellular matrix. Cathepsin K inhibition rescued the craniofacial phenotypes in the pheta1/2 double mutants. The abnormal renal and craniofacial phenotypes in the pheta1/2 mutant animals were consistent with the clinical presentation of a patient with a de novo arginine (R) to cysteine (C) variant (R6C) of PHETA1. Expressing the patient-specific variant in zebrafish exacerbated craniofacial deficits, suggesting that the R6C allele acts in a dominant-negative manner. Together, these results provide insights into the in vivo roles of PHETA1/2 and suggest that the R6C variant is contributory to the pathogenesis of disease in the patient.This article has an associated First Person interview with the first author of the paper.

Mutations in FAM50A suggest that Armfield XLID syndrome is a spliceosomopathy.

Intellectual disability (ID) is a heterogeneous clinical entity and includes an excess of males who harbor variants on the X-chromosome (XLID). We report rare FAM50A missense variants in the original Armfield XLID syndrome family localized in Xq28 and four additional unrelated males with overlapping features. Our fam50a knockout (KO) zebrafish model exhibits abnormal neurogenesis and craniofacial patterning, and in vivo complementation assays indicate that the patient-derived variants are hypomorphic. RNA sequencing analysis from fam50a KO zebrafish show dysregulation of the transcriptome, with augmented spliceosome mRNAs and depletion of transcripts involved in neurodevelopment. Zebrafish RNA-seq datasets show a preponderance of 3' alternative splicing events in fam50a KO, suggesting a role in the spliceosome C complex. These data are supported with transcriptomic signatures from cell lines derived from affected individuals and FAM50A protein-protein interaction data. In sum, Armfield XLID syndrome is a spliceosomopathy associated with aberrant mRNA processing during development.