cdon and boc affect trunk neural crest cell migration through a non-cell autonomous reduction of hedgehog signaling in zebrafish slow-twitch muscle

The immunoglobin superfamily members cdon and boc are transmembrane proteins implicated in regulating hedgehog signaling during vertebrate development. Recent work showing roles for these genes in axon guidance and neural crest cell migration further suggest that cdon/boc may play additional functions in regulating directed cell movements during development. Here we use novel and existing mutants to investigate a role for cdon and boc in zebrafish neural crest cell migration. We find that single cdon or boc mutant embryos exhibit normal neural crest phenotypes, but that neural crest migration is strikingly disrupted in double cdon/boc mutant embryos. We further show that this neural crest migration phenotype is associated with defects to the differentiation of slow-twitch muscle cells, and that this slow-twitch muscle phenotype is a consequence of reduced hedgehog signaling in mutant fish. While neural crest migratory ability is not affected in double mutant embryos, neural crest directionality is severely affected. These data suggest that neural crest migration defects are likely to be secondary to defects in slow-twitch muscle differentiation. Combined, our data add to a growing literature showing that cdon and boc act synergistically to promote hedgehog signaling during vertebrate development, and provide a foundation for using zebrafish to further study the function of these hedgehog receptor paralogs.

Zebrafish transposable elements show extensive diversification in age, genomic distribution, and developmental expression

There is considerable interest in understanding the effect of transposable elements (TEs) on embryonic development. Studies in humans and mice are limited by the difficulty of working with mammalian embryos, and by the relative scarcity of active TEs in these organisms. Zebrafish is an outstanding model for the study of vertebrate development and over half of its genome consists of diverse TEs. However, zebrafish TEs remain poorly characterized. Here we describe the demography and genomic distribution of zebrafish TEs and their expression throughout embryogenesis using bulk and single-cell RNA sequencing data. These results reveal a highly dynamic genomic ecosystem comprising nearly 2,000 distinct TE families, which vary in copy number by four orders of magnitude and span a wide range of ages. Longer retroelements tend to be retained in intergenic regions, whilst short interspersed nuclear elements (SINEs) and DNA transposons are more frequently found nearby or within genes. Locus-specific mapping of TE expression reveals extensive TE transcription during development. While two thirds of TE transcripts are likely driven by nearby gene promoters, we still observe stage and tissue-specific expression patterns in self-regulated TEs. Long terminal repeat (LTR) retroelements are most transcriptionally active immediately following zygotic genome activation, whereas DNA transposons are enriched amongst transcripts expressed in later stages of development. Single-cell analysis reveals several endogenous retroviruses expressed in specific somatic cell lineages. Overall, our study provides a valuable resource for using zebrafish as a model to study the impact of TEs on vertebrate development.

Runx1 protects against the pathological progression of osteoarthritis

Runt-related transcription factor-1 (Runx1) is required for chondrocyte-to-osteoblast lineage commitment by enhancing both chondrogenesis and osteogenesis during vertebrate development. However, the potential role of Runx1 in joint diseases is not well known. In the current study, we aimed to explore the role of Runx1 in osteoarthritis induced by anterior cruciate ligament transaction (ACLT) surgery. We showed that chondrocyte-specific Runx1 knockout (Runx1f/fCol2a1-Cre) aggravated cartilage destruction by accelerating the loss of proteoglycan and collagen II in early osteoarthritis. Moreover, we observed thinning and ossification of the growth plate, a decrease in chondrocyte proliferative capacity and the loss of bone matrix around the growth plate in late osteoarthritis. We overexpressed Runx1 by adeno-associated virus (AAV) in articular cartilage and identified its protective effect by slowing the destruction of osteoarthritis in cartilage in early osteoarthritis and alleviating the pathological progression of growth plate cartilage in late osteoarthritis. ChIP-seq analysis identified new targets that interacted with Runx1 in cartilage pathology, and we confirmed the direct interactions of these factors with Runx1 by ChIP-qPCR. This study helps us to understand the function of Runx1 in osteoarthritis and provides new clues for targeted osteoarthritis therapy.

Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model

Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development, as demonstrated by defects in the eye, brain, and skeleton that culminate in embryonic lethality when multiple HIPKs are lost in mice. While HIPKs are essential for development, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Because understanding the unique and shared functions of these essential proteins could directly benefit the fields of biology and medicine, we addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies our functional assessment. The single hipk present in the fly allowed us to perform rescue experiments with human HIPK genes that provide new insight into their individual functions not easily assessed in vertebrate models. Further, the abundance of genetic tools and established methods for monitoring specific developmental pathways and gross morphological changes in the fly allowed for functional comparisons in endogenous contexts. We first performed rescue experiments to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. We then showed the ability of each human HIPK to modulate Armadillo/β-catenin levels, JAK/STAT activity, proliferation, growth, and death, each of which have previously been described for Hipks, but never all together in comparable tissue contexts. Finally, we characterized novel developmental phenotypes induced by human HIPKs to gain insight to their unique functions. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs to determine their roles in a developmental context.

Authentication of a novel antibody to zebrafish collagen type XI alpha 1 chain (Col11a1a)

Objective Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

NudCL2 is an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis

Primary cilia extending from mother centrioles are essential for vertebrate development and homeostasis maintenance. Centriolar coiled-coil protein 110 (CP110) has been reported to suppress ciliogenesis initiation by capping the distal ends of mother centrioles. However, the mechanism underlying the specific degradation of mother centriole-capping CP110 to promote cilia initiation remains unknown. Here, we find that autophagy is crucial for CP110 degradation at mother centrioles after serum starvation in MEF cells. We further identify NudC-like protein 2 (NudCL2) as a novel selective autophagy receptor at mother centrioles, which contains an LC3-interacting region (LIR) motif mediating the association of CP110 and the autophagosome marker LC3. Knockout of NudCL2 induces defects in the removal of CP110 from mother centrioles and ciliogenesis, which are rescued by wild-type NudCL2 but not its LIR motif mutant. Knockdown of CP110 significantly attenuates ciliogenesis defects in NudCL2-deficient cells. In addition, NudCL2 morphants exhibit ciliation-related phenotypes in zebrafish, which are reversed by wild-type NudCL2, but not its LIR motif mutant. Importantly, CP110 depletion significantly reverses these ciliary phenotypes in NudCL2 morphants. Taken together, our data suggest that NudCL2 functions as an autophagy receptor mediating the selective degradation of mother centriole-capping CP110 to promote ciliogenesis, which is indispensable for embryo development in vertebrates.

Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l

Toll-like receptor 4 (Tlr) interactor with leucine-rich repeats (Tril) functions as a Tlr coreceptor to mediate innate immunity in adults. In Xenopus embryos, Tril triggers degradation of the transforming growth factor β (Tgf-ß) family inhibitor, Smad7. This enhances bone morphogenetic protein (Bmp) signaling to enable ventral mesoderm to commit to a blood fate. Here, we show that Tril simultaneously dampens Nodal signaling by catalytically activating the ubiquitin ligase NEDD4 Like (Nedd4l). Nedd4l then targets Nodal receptors for degradation. How Tril signals are transduced in a nonimmune context is unknown. We identify the ubiquitin ligase Pellino2 as a protein that binds to the cytoplasmic tail of Tril and subsequently forms a complex with Nedd4l and another E3 ligase, TNF-receptor associated factor 6 (Traf6). Pellino2 and Traf6 are essential for catalytic activation of Nedd4l, both in Xenopus and in mammalian cells. Traf6 ubiquitinates Nedd4l, which is then recruited to membrane compartments where activation occurs. Collectively, our findings reveal that Tril initiates a noncanonical Tlr-like signaling cascade to activate Nedd4l, thereby coordinately regulating the Bmp and Nodal arms of the Tgf-ß superfamily during vertebrate development.

Crosstalk between nitric oxide and retinoic acid pathways is essential for amphioxus pharynx development

During animal ontogenesis, body axis patterning is finely regulated by complex interactions among several signaling pathways. Nitric oxide (NO) and retinoic acid (RA) are potent morphogens that play a pivotal role in vertebrate development. Their involvement in axial patterning of the head and pharynx shows conserved features in the chordate phylum. Indeed, in the cephalochordate amphioxus, NO and RA are crucial for the correct development of pharyngeal structures. Here, we demonstrate the functional cooperation between NO and RA that occurs during amphioxus embryogenesis. During neurulation, NO modulates RA production through the transcriptional regulation of Aldh1a.2 that irreversibly converts retinaldehyde into RA. On the other hand, RA directly or indirectly regulates the transcription of Nos genes. This reciprocal regulation of NO and RA pathways is essential for the normal pharyngeal development in amphioxus and it could be conserved in vertebrates.