Ectodermal Patterning Research Articles

A novel mutation in the BTB domain impairs transcriptional repression function of KCTD1 leading to syndromic microtia

Microtia is a common birth defect affecting the external ears and encompasses a spectrum of congenital anomalies of the auricle. For some of the microtia-associated syndromes, the additional abnormalities are not easily observed or with variable expressivity. Identifying pathogenic mutations through genetic testing is of great help in recognizing these highly heterogeneous syndromes in clinical practice. We reported a novel de novo KCTD1 mutation in a Chinese patient with congenital microtia. It expands the mutational spectrum of KCTD1 and provide an additional scalp‐ear‐nipple patient with typical and atypical clinical presentations. The identified mutation in the BTB domain impairs the suppressive activity of the AP-2 transcription factor family and may impact on maintaining the finely tuned activity of WNT pathway, which directs stem cell development in ectoderm patterning and craniofacial development. Due to the variable expressive clinical phenotypes of syndromic microtia, genetic molecular testing could be of great help in the definite diagnosis.

ScRNA-seq analysis of cells comprising the amphioxus notochord

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

Embryonic organizer specification in the mud snail Ilyanassa obsoleta depends on intercellular signaling.

In early embryos of the caenogastropod snail Ilyanassa obsoleta, cytoplasmic segregation of a polar lobe is required for establishment of the D quadrant founder cell, empowering its great-granddaughter macromere 3D to act as a single-celled organizer that induces ectodermal pattern along the secondary body axis of the embryo. We present evidence that polar lobe inheritance is not sufficient to specify 3D potential, but rather makes the D macromere lineage responsive to some intercellular signal(s) required for normal expression of 3D-specific phenotypes. Experimental removal of multiple micromeres resulted in loss of organizer-linked MAPK activation, complete and specific defects of organizer-dependent larval organs, and progressive cell cycle retardation, leading to equalization of the normally accelerated division schedule of 3D (relative to the third-order macromeres of the A, B and C quadrants). Ablation of the second-quartet micromere 2d greatly potentiated the effects of first micromere quartet ablation. Our findings link organizer activation in I. obsoleta to the putative ancestral spiralian mechanism in which a signal from micromeres leads to specification of 3D among four initially equivalent macromeres.

Molecular evidence of anteroposterior patterning in adult echinoderms.

The origin of the pentaradial body plan of echinoderms from a bilateral ancestor is one of the most enduring zoological puzzles1,2. Because echinoderms are defined by morphological novelty, even the most basic axial comparisons with their bilaterian relatives are problematic. To revisit this classical question, we used conserved anteroposterior axial molecular markers to determine whether the highly derived adult body plan of echinoderms masks underlying patterning similarities with other deuterostomes. We investigated the expression of a suite of conserved transcription factors with well-established roles in the establishment of anteroposterior polarity in deuterostomes3-5 and other bilaterians6-8 using RNA tomography and in situ hybridization in the sea star Patiria miniata. The relative spatial expression of these markers in P. miniata ambulacral ectoderm shows similarity with other deuterostomes, with the midline of each ray representing the most anterior territory and the most lateral parts exhibiting a more posterior identity. Strikingly, there is no ectodermal territory in the sea star that expresses the characteristic bilaterian trunk genetic patterning programme. This finding suggests that from the perspective of ectoderm patterning, echinoderms are mostly head-like animals and provides a developmental rationale for the re-evaluation of the events that led to the evolution of the derived adult body plan of echinoderms.

The Foxi3 transcription factor is necessary for the fate restriction of placodal lineages at the neural plate border.

The Foxi3 transcription factor, expressed in the neural plate border at the end of gastrulation, is necessary for the formation of posterior placodes and is thus important for ectodermal patterning. We have created two knock-in mouse lines expressing GFP or a tamoxifen-inducible Cre recombinase to show that Foxi3 is one of the earliest genes to label the border between the neural tube and epidermis, and that Foxi3-expressing neural plate border progenitors contribute primarily to cranial placodes and epidermis from the onset of expression, but not to the neural crest or neural tube lineages. By simultaneously knocking out Foxi3 in neural plate border cells and following their fates, we show that neural plate border cells lacking Foxi3 contribute to all four lineages of the ectoderm - placodes, epidermis, crest and neural tube. We contrast Foxi3 with another neural plate border transcription factor, Zic5, the progenitors of which initially contribute broadly to all germ layers until gastrulation and gradually become restricted to the neural crest lineage and dorsal neural tube cells. Our study demonstrates that Foxi3 uniquely acts early at the neural plate border to restrict progenitors to a placodal and epidermal fate.

Maintenance of pluripotency-like signature in the entire ectoderm leads to neural crest stem cell potential

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like derivatives is obtained. Here, we monitor transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. We show maintenance of pluripotency-like signature (Nanog, Oct4/PouV, Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-like signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-like signature is found at multiple axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond “ectodermal-capacity” in chick and mouse embryos.

Molecular characterization of nervous system organization in the hemichordate acorn worm Saccoglossus kowalevskii.

Hemichordates are an important group for investigating the evolution of bilaterian nervous systems. As the closest chordate outgroup with a bilaterally symmetric adult body plan, hemichordates are particularly informative for exploring the origins of chordates. Despite the importance of hemichordate neuroanatomy for testing hypotheses on deuterostome and chordate evolution, adult hemichordate nervous systems have not been comprehensively described using molecular techniques, and classic histological descriptions disagree on basic aspects of nervous system organization. A molecular description of hemichordate nervous system organization is important for both anatomical comparisons across phyla and for attempts to understand how conserved gene regulatory programs for ectodermal patterning relate to morphological evolution in deep time. Here, we describe the basic organization of the adult hemichordate Saccoglossus kowalevskii nervous system using immunofluorescence, in situ hybridization, and transgenic reporters to visualize neurons, neuropil, and key neuronal cell types. Consistent with previous descriptions, we found the S. kowalevskii nervous system consists of a pervasive nerve plexus concentrated in the anterior, along with nerve cords on both the dorsal and ventral side. Neuronal cell types exhibited clear anteroposterior and dorsoventral regionalization in multiple areas of the body. We observed spatially demarcated expression patterns for many genes involved in synthesis or transport of neurotransmitters and neuropeptides but did not observe clear distinctions between putatively centralized and decentralized portions of the nervous system. The plexus shows regionalized structure and is consistent with the proboscis base as a major site for information processing rather than the dorsal nerve cord. In the trunk, there is a clear division of cell types between the dorsal and ventral cords, suggesting differences in function. The absence of neural processes crossing the basement membrane into muscle and extensive axonal varicosities suggest that volume transmission may play an important role in neural function. These data now facilitate more informed neural comparisons between hemichordates and other groups, contributing to broader debates on the origins and evolution of bilaterian nervous systems.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Lmo4a Contributes to Zebrafish Inner Ear and Vestibular Development via Regulation of the Bmp Pathway.

In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role. The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin. lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects. We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation.

Ndst1,aheparan sulfatemodificationenzyme,regulates neuroectodermal patterningbyenhancingWntsignalingin Xenopus.

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.

GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.

Feedback Regulation of Signaling Pathways for Precise Pre-Placodal Ectoderm Formation in Vertebrate Embryos.

Intracellular signaling pathways are essential to establish embryonic patterning, including embryonic axis formation. Ectodermal patterning is also governed by a series of morphogens. Four ectodermal regions are thought to be controlled by morphogen gradients, but some perturbations are expected to occur during dynamic morphogenetic movement. Therefore, a mechanism to define areas precisely and reproducibly in embryos, including feedback regulation of signaling pathways, is necessary. In this review, we outline ectoderm pattern formation and signaling pathways involved in the establishment of the pre-placodal ectoderm (PPE). We also provide an example of feedback regulation of signaling pathways for robust formation of the PPE, showing the importance of this regulation.

Human Pluripotent Stem Cell-Derived Micropatterned Ectoderm Allows Cell Sorting of Meso-Endoderm Lineages.

The human developmental processes during the early post-implantation stage instruct the specification and organization of the lineage progenitors into a body plan. These processes, which include patterning, cell sorting, and establishment of the three germ layers, have been classically studied in non-human model organisms and only recently, through micropatterning technology, in a human-specific context. Micropatterning technology has unveiled mechanisms during patterning and germ layer specification; however, cell sorting and their segregation in specific germ layer combinations have not been investigated yet in a human-specific in vitro system. Here, we developed an in vitro model of human ectodermal patterning, in which human pluripotent stem cells (hPSCs) self-organize to form a radially regionalized neural and non-central nervous system (CNS) ectoderm. We showed that by using micropatterning technology and by modulating BMP and WNT signals, we can regulate the appearance and spatial distribution of the different ectodermal populations. This pre-patterned ectoderm can be used to investigate the cell sorting behavior of hPSC-derived meso-endoderm cells, with an endoderm that segregates from the neural ectoderm. Thus, the combination of micro-technology with germ layer cross-mixing enables the study of cell sorting of different germ layers in a human context.

Gibbin mesodermal regulation patterns epithelial development.

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin,encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

The dual-specificity protein kinase Clk3 is essential for Xenopus neural development

During vertebrate development, the formation of the central nervous system (CNS) is initiated by neural induction and patterning of the embryonic ectoderm. We previously reported that Cdc2-like kinase 2 (Clk2) promotes neural development in Xenopus embryos by regulating morphogen signaling. However, the functions of other Clk family members and their roles in early embryonic development remain unknown. Here, we show that in addition to Clk2, Clk1 and Clk3 play a role in the formation of neural tissue in Xenopus. clk1 and clk3 are co-expressed in the developing neural tissue during early Xenopus embryogenesis. We found that overexpression of clk1 and clk3 increases the expression of neural marker genes in ectodermal explants. Furthermore, knockdown experiments showed that clk3 is required for the formation of neural tissues. These results suggest that Xenopus Clk3 plays an essential role in promoting neural development during early embryogenesis.

Post-transcriptional tuning of FGF signaling mediates neural crest induction

Ectodermal patterning is required for the establishment of multiple components of the vertebrate body plan. Previous studies have demonstrated that precise combinations of extracellular signals induce distinct ectodermal cell populations, such as the neural crest and the neural plate. Yet, we still lack understanding of how the response to inductive signals is modulated to generate the proper transcriptional output in target cells. Here we show that posttranscriptional attenuation of fibroblast growth factor (FGF) signaling is essential for the establishment of the neural crest territory. We found that neural crest progenitors display elevated expression of DICER, which promotes enhanced maturation of a set of cell-type-specific miRNAs. These miRNAs collectively target components of the FGF signaling pathway, a central player in the process of neural induction in amniotes. Inactivation of this posttranscriptional circuit results in a fate switch, in which neural crest cells are converted into progenitors of the central nervous system. Thus, the posttranscriptional attenuation of signaling systems is a prerequisite for proper segregation of ectodermal cell types. These findings demonstrate how posttranscriptional repression may alter the activity of signaling systems to generate distinct spatial domains of progenitor cells.

Temporal Modulations of NODAL, BMP, and WNT Signals Guide the Spatial Patterning in Self-Organized Human Ectoderm Tissues

Summary Developmental biology studies using model organisms suggest that the emergence of spatial patterning in the ectoderm is mediated by the morphogen gradients. However, it is still unclear whether the morphogen gradient is necessary and dominates the cell spatial patterning, particularly in human genetic background. Here, we demonstrate that human pluripotent stem cells can self-organize to concentric rings of all major cell types in ectoderm when cultured on micropatterned surfaces in a chemically defined condition. We reveal that modulating the dynamics of NODAL, BMP, and WNT signals is sufficient to control the spatial order of different cell types. Our mathematical model suggests that changes in wavelength and phase of signaling patterns formed via reaction-diffusion may be the mechanism by which temporal information is translated into spatial information. Together, our work demonstrates that in vitro human ectoderm microtissues have great potential in understanding the mechanisms of early-stage human development.

Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development.

Wnt/β-catenin signaling is an ancient pathway in metazoans and controls various developmental processes, in particular the establishment and patterning of the embryonic primary axis. In vertebrates, a graded Wnt activity from posterior to anterior endows cells with positional information in the central nervous system. Recent studies in hemichordates support a conserved role for Wnt/β-catenin in ectoderm antero-posterior patterning at the base of the deuterostomes. Ascidians are marine invertebrates and the closest relatives of vertebrates. By combining gain- and loss-of-function approaches, we have determined the role of Wnt/β-catenin in patterning the three ectoderm derivatives of the ascidian Ciona intestinalis, central nervous system, peripheral nervous system and epidermis. Activating Wnt/β-catenin signaling from gastrulation led to a dramatic transformation of the ectoderm with a loss of anterior identities and a reciprocal anterior extension of posterior identities, consistent with studies in other metazoans. Surprisingly, inhibiting Wnt signaling did not produce a reciprocal anteriorization of the embryo with a loss of more posterior identities like in vertebrates and hemichordate. Epidermis patterning was overall unchanged. Only the identity of two discrete regions of the central nervous system, the anteriormost and the posteriormost regions, were under the control of Wnt. Finally, the caudal peripheral nervous system, while being initially Wnt dependent, formed normally. Our results show that the Ciona embryonic ectoderm responds to Wnt activation in a manner that is compatible with the proposed function for this pathway at the base of the deuterostomes. However, possibly because of its fast and divergent mode of development that includes extensive use of maternal determinants, the overall antero-posterior patterning of the Ciona ectoderm is Wnt independent, and Wnt/β-catenin signaling controls the formation of some sub-domains. Our results thus indicate that there has likely been a drift in the developmental systems controlling ectoderm patterning in the lineage leading to ascidians.

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm.

During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight into this process; however, there are currently no systems in which to study ectodermal patterning in humans. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning, in which human embryonic stem cells self-organize to form robust and quantitatively reproducible patterns corresponding to the complete medial-lateral axis of the embryonic ectoderm. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation. Thus, our platform is a powerful tool for studying human ectoderm patterning and for improving directed differentiation protocols.This article has an associated 'The people behind the papers' interview.

Fam46a regulates BMP-dependent pre-placodal ectoderm differentiation in Xenopus.

The pre-placodal ectoderm (PPE) is a specialized ectodermal region which gives rise to the sensory organs and other systems. The PPE is induced from the neural plate border during neurulation, but the molecular mechanism of PPE formation is not fully understood. In this study, we examined the role of a newly identified PPE gene, Fam46a, during embryogenesis. Fam46a contains a nucleoside triphosphate transferase domain, but its function in early development was previously unclear. We show that Fam46a is expressed in the PPE in Xenopus embryos, and Fam46a knockdown induces abnormalities in the eye formation and the body color. At the neurula stage, Fam46a upregulates the expression of PPE genes and inhibits neural crest formation. We also show that Fam46a physically interacts with Smad1/Smad4 and positively regulates BMP signaling. From these results, we conclude that Fam46a is required for PPE formation via the positive regulation of BMP signaling. Our study provides a new mechanism of ectodermal patterning via cell-autonomous regulation of BMP signaling in the PPE.