Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Botryllus schlosseri, a marine colonial chordate, has produced valuable insight in the study of tissue regeneration (1, 2), allorecognition (3–6), and stem cells (7, 8). B. schlosseri can be developed as a chordate following sexual reproduction, which gives rise to a tadpole that hatches and finds a subtidal surface to attach to, before metamorphosis into a sessile invertebrate. The metamorphosed individual develops 1 to 3 buds (covered by a common gelatinous tunic) that develop the invertebrate body plan, and several individuals anastomose extracorporeal blood vessels in the tunic to form a colony. Every week this colonial organism undergoes a de novo robust regeneration mediated by adult stem cells that participate in the formation of all body organs, including the central nervous system. The lifespan of B. schlosseri is plastic and amenable to change. Wild colonies are characterized by short lifespans ranging from several months to a few years (9–12) whereas colonies grown in the laboratory can reach 20 y of age (12–17). Systemic changes occur over time in older colonies, including a slower heartbeat, reduced zooid size, decreased regenerative capacity, a shift in cellular composition (e.g., higher level of engulfing phagocytic, cytotoxic, and pigment cells), and shifting patterns of cyclic gene expression associated with aging (17). Despite the key evolutionary position of B. schlosseri as a member of a clade considered to be the sister group of vertebrates (the body plan of the sexually reproduced larval tadpole is along the architecture of most chordate fetuses) (18), and previous literature describing its central nervous system (19–22), little is known about changes in brain morphology, colony behavior, and gene expression associated with the regular, cyclical processes of neural generation and degeneration and how they vary with colony age.Here we introduce B. schlosseri as a model for evolutionary neuroscience. The B. schlosseri genome has been sequenced (5) and an atlas of the molecular and morphological signatures of each developmental stage has been previously generated using microscopy and RNA sequencing (RNA-seq) (22). B. schlosseri can reproduce either sexually through embryogenesis or asexually through blastogenesis. The sexually produced larvae develop two brains (a functional larval brain and the rudiment of the adult brain), a sensory organ detecting light and gravity, a notochord, and a dorsal nerve cord. The larval brain (along with the notochord, segmented musculature, and tail) is absorbed during the settlement and metamorphosis which precedes colony formation. As a sedentary organism, B. schlosseri buds new individuals that together live as a colony composed of individual zooids. Each zooid reproduces asexually in weekly, stem cell-mediated budding cycles (7). Budding occurs during this cycle when, simultaneously throughout the colony, a new generation of buds begin to develop into adult individuals. During budding, all organs including the brain, are created anew without passing through a larval (embryonic and fetal) stage. The life of adult zooids lasts ∼1 wk after which their bodies undergo a synchronized wave of programmed cell death and phagocytic removal called takeover (i.e., TO) (16, 23) in which the nervous systems of old zooids degenerate in tangent to brain formation in the young buds.In this study, we describe 2 different pathways of neurodegeneration that occur during the life cycle of the colonial chordate B. schlosseri. The first occurs every week as a part of a regenerative developmental cycle that occurs independent of age; the second is associated with colony aging. We integrate neural imaging (transmission electron microscopy [TEM] and confocal), three-dimensional (3D) reconstructions, behavioral assay, and RNA-seq of enriched brains (Fig. 1) to study morphological and molecular changes in brains of adult B. schlosseri across developmental stages and ages as associated with changes in zooid behavior. In providing evidence of the cellular and molecular linkages between each neurodegenerative process, we document an unanticipated degree of similarity between the 2 that may be useful in identifying the evolutionary basis of the pathological mechanisms responsible for age-induced loss of neuron structure and function in other phylogeny. Indeed, we argue that given 1) a unique and assayable life cycle, 2) real-time monitoring enabled by transparent body and vasculature, 3) adult tissue specific stem cells that can be enriched and transplanted, 4) morphological differences associated with colony age, and 5) a previously sequenced genome; B. schlosseri provides a novel and valuable platform for the study of neurogenesis, neurodegeneration, and evolutionary neuroscience.Open in a separate windowFig. 1.Sampling and methods used to characterize weekly cycles of neurodegeneration in B. schlosseri colonies. Schematic depicting the various methods employed over the course of the investigation including neural complex dissection, confocal staining, histology, TEM, behavioral experiments and the intervals over which neural complex sampling was conducted (over the weekly cycle and with age in adult individuals). Genetically identical subclones prepared via incision and separation were used in diverse experimental groups.