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Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1, 2, 3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia. acceptedVersion

Excretion is an essential physiological process, carried out by all living organisms regardless of their size or complexity(1–3). Most animals, which include both protostomes (e.g. flies, flatworms) and deuterostomes (e.g. humans, sea urchins) (together Nephrozoa(4, 5)), possess specialized excretory organs. Those organs exhibit an astonishing diversity, ranging from units composed of just three distinct cells (e.g. protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g. vertebrate kidneys)(6, 7). Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported(8, 9), the molecular development of nephrozoan excretory organs has never been systematically studied in a comparative context(6). Here we show that a set of highly conserved transcription factors and structural proteins is expressed during the development of excretory organs of six species that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity witnessed in the vertebrate kidney and flatworm protonephridia(8) is also seen in the developing excretory organs of other Nephrozoa. In addition, orthologous structural proteins forming the ultrafiltration apparatus are expressed in all these organs in the filter-forming cells. Our results strongly suggest that excretory organs are homologous and are patterned by the conserved set of developmental genes. We propose that the last common nephrozoan ancestor possessed an ultrafiltration-based, ciliated excretory organ, a structure that later gave rise to the vast diversity of extant excretory organs, including the human kidney.Significance statementMost of the bilaterally symmetrical animals excrete through specialized excretory organs, such as kidneys and nephridia. However, due to the morphological diversity of these organs, it remains unknown whether those structures evolved from a common ancestral organ or appeared several times independently during evolution. In order to answer the question about the origin of excretory organs we investigated the molecular pathways and structural genes involved in the development of nephridia in 6 animal species representing major evolutionary lineages. We show that diverse excretory organs share an ancient molecular patterning and structural molecules. Our results provide strong evidence that all excretory organs originated from a single, simple organ that performed urine production by ultrafiltration in deep geological past.

Hemocytes are immune cells in the hemolymph of invertebrates that play multiple roles in response to stressors; hemocyte mortality can thus serve as an indicator of overall animal health. However, previous research has often analyzed hemolymph samples pooled from several individuals, which precludes tracking individual responses to stressors over time. The ability to track individuals is important, however, because large inter-individual variation in response to stressors can confound the interpretation of pooled samples. Here, we describe protocols for analysis of inter- and intra-individual variability in hemocyte mortality across repeated hemolymph samples of California mussels, Mytilus californianus, free from typical abiotic stressors. To assess individual variability in hemocyte mortality with serial sampling, we created four groups of 15 mussels each that were repeatedly sampled four times: at baseline (time zero) and three subsequent times separated by either 24, 48, 72, or 168 h. Hemocyte mortality was assessed by fluorescence-activated cell sorting (FACS) of cells stained with propidium iodide. Our study demonstrates that hemolymph can be repeatedly sampled from individual mussels without mortality; however, there is substantial inter- and intra-individual variability in hemocyte mortality through time that is partially dependent on the sampling interval. Across repeated samples, individual mussels' hemocyte mortality had, on average, a range of ~6% and a standard deviation of ~3%, which was minimized with sampling periods ≥72 h apart. Due to this intra-individual variability, obtaining ≥2 samples from a specimen will more accurately establish an individual's baseline. Pooled-sample means were similar to individual-sample means; however, pooled samples masked the individual variation in each group. Overall, these data lay the foundation for future work exploring individual mussels' temporal responses to various stressors on a cellular level.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventral-most regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin1 (Edn1) signaling in the intermediate joint-forming region yet a surprisingly minor role in ventral-most regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of coregulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then performed mutational analysis of a number of these genes, which uncovered antagonistic functions of two new Edn1 targets,follistatin a(fsta) andemx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Ednl and points to novel candidates for craniofacial disorders.Summary StatementUsing zebrafish to purify distinct groups of embryonic cells, Askary et al. have created a detailed map of how thousands of genes are deployed to shape the developing face.

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways − Jagged-Notch, Endothelin1 (Edn1), and Bmp − regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face., Author Summary The exquisite functions of the vertebrate face require the precise formation of its underlying bones. Remarkably, many of the genes required to shape the facial skeleton are the same from fish to man. In this study, we use the powerful zebrafish system to understand how the skeletal components of the face acquire different shapes during development. To do so, we analyze a series of mutants that disrupt patterning of the facial skeleton, and then assess how the genes affected in these mutants control cell fate in skeletal progenitor cells. From these genetic studies, we found that several pathways converge to control when and where progenitor cells commit to a cartilage fate, thus controlling the size and shape of cartilage templates for the later-arising bones. Our work thus reveals how regulating the timing of when progenitor cells make skeleton helps to shape the bones of the zebrafish face. As mutations in many of the genes studied are implicated in human craniofacial defects, differences in the timing of progenitor cell differentiation may also explain the wonderful diversity of human faces.