Embryo-scale reverse genetics at single-cell resolution.

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1-4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

The ion channel Anoctamin 10/TMEM16K coordinates organ morphogenesis across scales in the urochordate notochord.

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.

Comprehensive single-cell transcriptome lineages of a proto-vertebrate.

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development-from the onset of gastrulation to swimming tadpoles-in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

Hinge point emergence in mammalian spinal neurulation.

Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hinge point, then both the median hinge point and dorsolateral hinge points, followed by dorsolateral hinge points only. The biomechanical mechanism of hinge point formation in the mammalian neural tube is poorly understood. Here we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, nonneural ectoderm expansion, and neural plate adhesion to the notochord. Dorsolateral hinge points emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hinge point formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

The embryonic node behaves as an instructive stem cell niche for axial elongation.

In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.

Single-cell morphometrics reveals ancestral principles of notochord development.

Embryonic tissues are shaped by the dynamic behaviours of their constituent cells. To understand such cell behaviours and how they evolved, new approaches are needed to map out morphogenesis across different organisms. Here, we apply a quantitative approach to learn how the notochord forms during the development of amphioxus: a basally branching chordate. Using a single-cell morphometrics pipeline, we quantify the geometries of thousands of amphioxus notochord cells, and project them into a common mathematical space, termed morphospace. In morphospace, notochord cells disperse into branching trajectories of cell shape change, revealing a dynamic interplay between cell shape change and growth that collectively contributes to tissue elongation. By spatially mapping these trajectories, we identify conspicuous regional variation, both in developmental timing and trajectory topology. Finally, we show experimentally that, unlike ascidians but like vertebrates, posterior cell division is required in amphioxus to generate full notochord length, thereby suggesting this might be an ancestral chordate trait that is secondarily lost in ascidians. Altogether, our novel approach reveals that an unexpectedly complex scheme of notochord morphogenesis might have been present in the first chordates. This article has an associated 'The people behind the papers' interview.

Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation.

Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.

Development of a straight vertebrate body axis.

The vertebrate body plan is characterized by the presence of a segmented spine along its main axis. Here, we examine the current understanding of how the axial tissues that are formed during embryonic development give rise to the adult spine and summarize recent advances in the field, largely focused on recent studies in zebrafish, with comparisons to amniotes where appropriate. We discuss recent work illuminating the genetics and biological mechanisms mediating extension and straightening of the body axis during development, and highlight open questions. We specifically focus on the processes of notochord development and cerebrospinal fluid physiology, and how defects in those processes may lead to scoliosis.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning.

The elongating mouse anteroposterior axis is supplied by progenitors with distinct tissue fates. It is not known whether these progenitors confer anteroposterior pattern to the embryo. We have analysed the progenitor population transcriptomes in the mouse primitive streak and tail bud throughout axial elongation. Transcriptomic signatures distinguish three known progenitor types (neuromesodermal, lateral/paraxial mesoderm and notochord progenitors; NMPs, LPMPs and NotoPs). Both NMP and LPMP transcriptomes change extensively over time. In particular, NMPs upregulate Wnt, Fgf and Notch signalling components, and many Hox genes as progenitors transit from production of the trunk to the tail and expand in number. In contrast, the transcriptome of NotoPs is stable throughout axial elongation and they are required for normal axis elongation. These results suggest that NotoPs act as a progenitor niche whereas anteroposterior patterning originates within NMPs and LPMPs.

Development and regeneration dynamics of the Medaka notochord.

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon, a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon â€‹+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo, and provides new insights in its development and regeneration properties.

Direct activation of chordoblasts by retinoic acid is required for segmented centra mineralization during zebrafish spine development.

Zebrafish mutants with increased retinoic acid (RA) signaling due to the loss of the RA-inactivating enzyme Cyp26b1 develop a hyper-mineralized spine with gradually fusing vertebral body precursors (centra). However, the underlying cellular mechanisms remain incompletely understood. Here, we show that cells of the notochord epithelium named chordoblasts are sensitive to RA signaling. Chordoblasts are uniformly distributed along the anteroposterior axis and initially generate the continuous collagenous notochord sheath. However, subsequently and iteratively, subsets of these cells undergo further RA-dependent differentiation steps, acquire a stellate-like shape, downregulate expression of the collagen gene col2a1a, switch on cyp26b1 expression and trigger metameric sheath mineralization. This mineralization fails to appear upon chordoblast-specific cell ablation or RA signal transduction blockade. Together, our data reveal that, despite their different developmental origins, the activities and regulation of chordoblasts are very similar to those of osteoblasts, including their RA-induced transition from osteoid-producing cells to osteoid-mineralizing ones. Furthermore, our data point to a requirement for locally controlled RA activity within the chordoblast layer in order to generate the segmented vertebral column.

14-3-3εa directs the pulsatile transport of basal factors toward the apical domain for lumen growth in tubulogenesis.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Patterns of brachyury expression in chordomas.

INTRODUCTION: Chordomas are rare malignant midline tumors, presumed to arise from notochordal remnants. This was further suggested by the discovery of the brachyury in chordomas pathogenesis. Its immunohistochemical expression has become the principal adjunct in the diagnosis of chordomas. However, studies about brachyury expression in chordomas are not fully comparable, mainly because they use different primary antibodies. Thus, the aim of this study is to investigate the expression of brachyury expression in a series of chordomas in conjunction to clinicopathological characteristics and to review the relevant literature providing all the details needed in the immunohistochemical study of brachyury. MATERIALS AND METHODS: This is a retrospective study of 62 chordomas, diagnosed over a 22-year period. No dedifferentiated or poorly differentiated cases were included. A monoclonal primary antibody (clone A-4) was used and brachyury expression was evaluated by the H-score. Clinicopathological parameters studied were age, sex, tumor localization, decalcification status and tissue age. Fetal notochords were used for comparison. RESULTS: Mean H-score of nuclear brachyury expression was 129.8. The tissue age significantly influenced brachyury expression, the older samples expressing less brachyury. Decalcification demonstrated a trend to weaken brachyury expression. Clinical characteristics were not correlated with the patterns of brachyury expression. Notochords were negative. Literature review reveals several polyclonal antibodies used and a positivity of 75%-100% in chordomas with even more variable results in notochords. CONCLUSION: In chordomas, as in other tumor types, an uniformization of studies about brachyury expression is needed, by considering the clone used, and the decalcification and the age of the sample, given the growing importance of brachyury in diagnosis and therapeutic steps.

Multiple inputs into a posterior-specific regulatory network in the Ciona notochord.

The gene regulatory networks underlying Ciona notochord fate specification and differentiation have been extensively investigated, but the regulatory basis for regionalized expression within the notochord is not understood. Here we identify three notochord-expressed genes, C11.331, C12.115 and C8.891, with strongly enriched expression in the secondary notochord cells at the posterior tip of the tail. C11.331 and C12.115 share a distinctive expression pattern that is highly enriched in the secondary notochord lineage but also graded within that lineage with the strongest expression at the posterior tip. Both genes show similar responses to pharmacological perturbations of Wnt and FGF signaling, consistent with an important role for Wnt and FGF ligands expressed at the tail tip. Reporter analysis indicates that the C11.331 cis-regulatory regions are extensively distributed, with multiple non-overlapping regions conferring posterior notochord-enriched expression. Fine-scale analysis of a minimal cis-regulatory module identifies discrete positive and negative elements including a strong silencer. Truncation of the silencer region leads to increased expression in the primary notochord, indicating that C11.331 expression is influenced by putative regulators of primary versus secondary notochord fate. The minimal CRM contains predicted ETS, GATA, LMX and Myb sites, all of which lead to reduced expression in secondary notochord when mutated. These results show that the posterior-enriched notochord expression of C11.331 depends on multiple inputs, including Wnt and FGF signals from the tip of the tail, multiple notochord-specific regulators, and yet-to-be identified regulators of regional identity within the notochord.

Ascidian notochord elongation.

The elongation of embryo and tissue is a key morphogenetic event in embryogenesis and organogenesis. Notochord, a typical chordate organ, undergoes elongation to perform its regulatory roles and to form the structural support in the embryo. Notochord elongation is morphologically similar across all chordates, but ascidian has evolved distinct molecular and cellular processes. Here, we summarize the current understanding of ascidian notochord elongation. We divide the process into three phases and discuss the underlying molecular mechanisms in each phase. In the first phase, the notochord converges and extends through invagination and mediolateral intercalation, and partially elongates to form a single diameter cell column along the anterior-posterior axis. In the second phase, a cytokinesis-like actomyosin ring is constructed at the equator of each cell and drives notochord to elongate approximately two-fold. The molecular composition and architecture of the ascidian notochord contractile ring are similar to that of the cytokinetic ring. However, the notochord contractile ring does not impose cell division but only drives cell elongation followed by disassembly. We discuss the self-organizing property of the circumferential actomyosin ring, and why it disassembles when certain notochord length is achieved. The similar ring structures are also present in the elongation process of other organs in evolutionarily divergent animals such as Drosophila and C. elegans. We hereby propose that actomyosin ring-based circumferential contraction is a common mechanism adopted in diverse systems to drive embryo and tissue elongation. In the third phase, the notochord experiences tubulogenesis and the endothelial-like cells crawl bi-directionally on the notochord sheath to further lengthen the notochord. In this review, we also discuss extracellular matrix proteins, notochord sheath, and surrounding tissues that may contribute to notochord integrity and morphogenesis.

Developmental atlas of appendicularian Oikopleura dioica actins provides new insights into the evolution of the notochord and the cardio-paraxial muscle in chordates.

Locomotion by tail beating powered by a system of bilateral paraxial muscle and notochord is likely one of the key evolutionary innovations that facilitated the origin and radiation of chordates. The innovation of paraxial muscle was accompanied by gene duplications in stem chordates that gave rise to muscular actins from cytoplasmic ancestral forms, which acquired contractile capability thanks to the recruitment of the myosin motor-machinery. To better understand the role of actin diversification during the evolution of chordates, in this work we have characterized the complete actin catalogue of the appendicularian Oikopleura dioica, an urochordate that maintains a chordate body plan throughout its life, including the notochord in a muscled tail that confers an active free-living pelagic style. Our genomic survey, phylogenetic analyses and Diagnostic-Actin-Values (DAVs) reveal that O. dioica has four muscular actins (ActnM1-4) and three cytoplasmic actins (ActnC1-3), most of which originated by independent gene duplications during the evolution of the appendicularian lineage. Detailed developmental expression atlas of the complete actin catalogue of O. dioica reveals differences in the temporal-regulation and tissue-specificity of different actin paralogs, suggesting complex processes of subfunctionalization during the evolution of urochordates. Our results suggest the presence of a "cardio-paraxial" muscular actin at least in the last common ancestor of Olfactores (i.e. vertebrates+urochordates). Our results reveal highly dynamic tissue-specific expression patterns for some cytoplasmic actins, including the notochord, ciliated cells and neurons with axonal projections, which challenge the classic housekeeping notion ascribed to these genes. Considering that previous work had demonstrated the existence of notochord-specific actins in cephalochordates, the tissue-specific expression of two cytoplasmic actins in the notochord of O. dioica suggests that this pattern plausibly reflects the ancestral condition of chordates, and provides new insights to better understand the evolutionary origin of the notochord.

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.

The Ciona myogenic regulatory factor functions as a typical MRF but possesses a novel N-terminus that is essential for activity.

Electroporation-based assays were used to test whether the myogenic regulatory factor (MRF) of Ciona intestinalis (CiMRF) interferes with endogenous developmental programs, and to evaluate the importance of its unusual N-terminus for muscle development. We found that CiMRF suppresses both notochord and endoderm development when it is expressed in these tissues by a mechanism that may involve activation of muscle-specific microRNAs. Because these results add to a large body of evidence demonstrating the exceptionally high degree of functional conservation among MRFs, we were surprised to discover that non-ascidian MRFs were not myogenic in Ciona unless they formed part of a chimeric protein containing the CiMRF N-terminus. Equally surprising, we found that despite their widely differing primary sequences, the N-termini of MRFs of other ascidian species could form chimeric MRFs that were also myogenic in Ciona. This domain did not rescue the activity of a Brachyury protein whose transcriptional activation domain had been deleted, and so does not appear to constitute such a domain. Our results indicate that ascidians have previously unrecognized and potentially novel requirements for MRF-directed myogenesis. Moreover, they provide the first example of a domain that is essential to the core function of an important family of gene regulatory proteins, one that, to date, has been found in only a single branch of the family.