Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Positioning a multifunctional basic helix-loop-helix transcription factor within the Ciona notochord gene regulatory network.

In a multitude of organisms, transcription factors of the basic helix-loop-helix (bHLH) family control the expression of genes required for organ development and tissue differentiation. The functions of different bHLH transcription factors in the specification of nervous system and paraxial mesoderm have been widely investigated in various model systems. Conversely, the knowledge of the role of these regulators in the development of the axial mesoderm, the embryonic territory that gives rise to the notochord, and the identities of their target genes, remain still fragmentary. Here we investigated the transcriptional regulation and target genes of Bhlh-tun1, a bHLH transcription factor expressed in the developing Ciona notochord as well as in additional embryonic territories that contribute to the formation of both larval and adult structures. We describe its possible role in notochord formation, its relationship with the key notochord transcription factor Brachyury, and suggest molecular mechanisms through which Bhlh-tun1 controls the spatial and temporal expression of its effectors.

Investigating Evolutionarily Conserved Molecular Mechanisms Controlling Gene Expression in the Notochord.

Ascidian embryos have been employed as model systems for studies of developmental biology for well over a century, owing to their desirable blend of experimental advantages, which include their rapid development, traceable cell lineage, and evolutionarily conserved morphogenetic movements. Two decades ago, the development of a streamlined electroporation method drastically reduced the time and cost of transgenic experiments, and, along with the elucidation of the complete genomic sequences of several ascidian species, propelled these simple chordates to the forefront of the model organisms available for studies of regulation of gene expression. Numerous ascidian sequences with tissue-specific enhancer activity were isolated and rapidly characterized through systematic in vivo experiments that would require several weeks in most other model systems. These cis-regulatory sequences include a large collection of notochord enhancers, which have been used to visualize notochord development in vivo, to generate mutant phenotypes, and to knock down genes of interest. Moreover, their detailed characterization has allowed the reconstruction of different branches of the notochord gene regulatory network. This chapter describes how the use of transgenic techniques has rendered the ascidian Ciona a competitive model organism for studies of notochord development, evolution, and gene regulation.

Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis.

The cilium both releases and binds to extracellular vesicles (EVs). EVs may be used by cells as a form of intercellular communication and mediate a broad range of physiological and pathological processes. The mammalian polycystins (PCs) localize to cilia, as well as to urinary EVs released from renal epithelial cells. PC ciliary trafficking defects may be an underlying cause of autosomal dominant polycystic kidney disease (PKD), and ciliary-EV interactions have been proposed to play a central role in the biology of PKD. In Caenorhabditis elegans and mammals, PC1 and PC2 act in the same genetic pathway, act in a sensory capacity, localize to cilia, and are contained in secreted EVs, suggesting ancient conservation. However, the relationship between cilia and EVs and the mechanisms generating PC-containing EVs remain an enigma. In a forward genetic screen for regulators of C. elegans PKD-2 ciliary localization, we identified CIL-7, a myristoylated protein that regulates EV biogenesis. Loss of CIL-7 results in male mating behavioral defects, excessive accumulation of EVs in the lumen of the cephalic sensory organ, and failure to release PKD-2::GFP-containing EVs to the environment. Fatty acylation, such as myristoylation and palmitoylation, targets proteins to cilia and flagella. The CIL-7 myristoylation motif is essential for CIL-7 function and for targeting CIL-7 to EVs. C. elegans is a powerful model with which to study ciliary EV biogenesis in vivo and identify cis-targeting motifs such as myristoylation that are necessary for EV-cargo association and function.