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Histological and morphological analysis of eye development in Mab21l1-deficient mice. Gross appearance of Mab21l1 +/– (A) and Mab21l1 –/– (B) eyes. The Mab21l1 +/– lens is well developed (A; P1, E16) and pigmented retinal epithelium is almost round, whereas the Mab21l1 –/– eye has no lens and the retinal pigmented epithelium is abnormally indented (B; P1, E16, E12.5). No difference is evident at E10.5. HE-stained coronal sections of E17 (C,D), E13 (E,F), E10.5 (G,H) and E9.5 (I,J) embryos. At E17 and E13, no lens is evident in the Mab21l1 –/– eye (D,F). Brackets (C,D) indicate the thickness of the retina, arrowheads (D) indicate abnormal corneal swelling and the arrow (D) indicates the optic nerve. (H) At E10.5, a malformed lens placode is evident in the Mab21l1 –/– eye (arrowheads). (I,J) At E9.5, little difference is evident between Mab21l1 +/– (I) and Mab21l1 –/– (J) eyes. The presumptive lens placode is abnormally thin and narrow in Mab21l1 –/– embryos (J; brackets) compared with Mab21l1 +/– embryos (I; brackets). Scale bars: 200 µm for C,D; 100 µm for E,F; 50 µm for G-J.
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The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologs, Mab21l1 and Mab21l2, have been identified in many species. We describe the generation of Mab21l1-defi...
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... As the field of in silico gene regulatory network prediction expands, this approach can be applied to other factors, e.g. FOXE3, HSF4, MAB21L1, PITX3, TFAP2A, etc., for which loss-of-function gene expression datasets exist and can be used as validation (Blixt et al., 2000;Brownell et al., 2000;Fujimoto et al., 2004;Ho et al., 2009;Min et al., 2004;West-Mays et al., 1999;Yamada et al., 2003). We intend that this study should set a foundation for future in-depth interrogations of lens development at single-cell resolution. ...
Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation.
... Recent work has shown that cGAS and related animal proteins, the cGAS-like Receptors (cGLRs), are present in nearly all metazoan taxa and generate diverse cyclic dinucleotide signals [45]. However, the immune functions of Mab21L1 and MB21D2 remain unclear, although Mab21L1 has been shown to be important for development [46][47][48]. ...
Animals use a variety of cell-autonomous innate immune proteins to detect viral infections and prevent replication. Recent studies have discovered that a subset of mammalian antiviral proteins have homology to antiphage defense proteins in bacteria, implying that there are aspects of innate immunity that are shared across the Tree of Life. While the majority of these studies have focused on characterizing the diversity and biochemical functions of the bacterial proteins, the evolutionary relationships between animal and bacterial proteins are less clear. This ambiguity is partly due to the long evolutionary distances separating animal and bacterial proteins, which obscures their relationships. Here, we tackle this problem for 3 innate immune families (CD-NTases [including cGAS], STINGs, and viperins) by deeply sampling protein diversity across eukaryotes. We find that viperins and OAS family CD-NTases are ancient immune proteins, likely inherited since the earliest eukaryotes first arose. In contrast, we find other immune proteins that were acquired via at least 4 independent events of horizontal gene transfer (HGT) from bacteria. Two of these events allowed algae to acquire new bacterial viperins, while 2 more HGT events gave rise to distinct superfamilies of eukaryotic CD-NTases: the cGLR superfamily (containing cGAS) that has diversified via a series of animal-specific duplications and a previously undefined eSMODS superfamily, which more closely resembles bacterial CD-NTases. Finally, we found that cGAS and STING proteins have substantially different histories, with STING protein domains undergoing convergent domain shuffling in bacteria and eukaryotes. Overall, our findings paint a picture of eukaryotic innate immunity as highly dynamic, where eukaryotes build upon their ancient antiviral repertoires through the reuse of protein domains and by repeatedly sampling a rich reservoir of bacterial antiphage genes.
... The advent of systems-level multiomic approaches, such as combined snRNA/snATAC-seq, stand to better refine these predictions by establishing complementary layers of evidence that can reinforce the observed regulatory relationships. As the field of insilico prediction of gene regulatory networks expands, this predictive approach can be applied to other factors, e.g., FOXE3, HSF4, MAB21L1, PITX3, TFAP2A, etc., for which loss-of-function global gene expression datasets have been generated and thus can be used to validate predictions[102][103][104][105][106][107][108] . ...
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... Previous structural analysis of Mab21L1-related proteins confirms homology with cGLR enzymes in innate immunity, 28 but Mab21L1-related proteins have a non-immune role in developmental tissue patterning, and it is not understood if these proteins synthesize nucleotide second messenger signals. [29][30][31][32] Notably, some specific metazoan phyla such as nematodes and most platyhelminthes encode no predicted cGLRs other than Mab21-like proteins, suggesting that innate immune cGLR signaling pathways have been specifically lost in select metazoan lineages similar to genetic loss events observed for TLRs in rotifera and platyhelminthes and NLRs in Drosophila and C. elegans. 9,10,33,34 Analysis of cGLR sequences reveals specific patterns of evolution in animal immunity and protein features distinct from bacterial CD-NTase anti-phage defense enzymes. ...
Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.
... 3D FSC values were calculated by the Remote 3DFSC Processing Server (https://3dfsc.salk. edu)73 . EMRinger scores were calculated by EMRinger63 . ...
In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP–AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP–AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling¹. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases² (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers³. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2–CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase–target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.
... We also tested if foxe3 is rescued by mab21l1 injections (Fig. 8C) because its 378 expression is reduced in the murine mab21l1 mutant (Yamada et al., 2003). As opposed to 379 cryg1, a complete loss of foxe3 expression has already occurred by st.24 in the six3 mutant 380 We next examined if BMP activity could be rescued by mab21l1 mRNA injection (Fig. 384 8D). ...
... We also found signaling networks are down regulated by reductions in nuclear proteins 428 mab21l1 and mab21l2 in the six3 mutant PR, both of which were shown as well to cause lens 429 defects in knockout mice (Yamada et al., 2003;Yamada et al., 2004). Importantly, Mab21l2 is 430 known to cause a PR defect leading to lens defects (Yamada et al., 2004), which is consistent 431 with our hypothesis that failure of the PR in frog six3 mutant causes the lens defect seen here. ...
... By contrast, Mab21l1 is suggested to function in the PLE cell-autonomously for lens formation; 433 however, it is worth pointing out that the mutant mice also have abnormal retinal development 434 (Yamada et al., 2003), possibly augmenting the failure of lens formation non-autonomously. Our 435 data shows that mab21l1 mRNA could partially rescue lens formation in the six3 mutant as 436 evidenced by foxe3 and cryg1 expression and coincident with recovery of BMP activity as 437 evidenced by smad7 expression, a readout of activation of BMP pathway signaling (Fig. 8). ...
The Six3 transcription factor is essential for forebrain and eye development, and SIX3 mutations cause the congenital disorder holoprosencephaly. We created a six3 mutant in Xenopus tropicalis with a mild holoprosencephaly phenotype, and unlike mouse Six3 mutants that are headless/eyeless, the Xenopus mutant forms some eye structures, allowing direct study of Six3 function in eye formation. We focus here on striking deficits in lens formation. Early lens induction occurs normally in the mutant, e.g., the essential eye gene pax6 , is activated in lens ectoderm, persisting in the eye to a late developmental stage, but in many embryos the lens fails to form. We found that bmp4, bmp7 . 1, smad7, dll1, dlc, mab21l1 and/or mab21l2 , previously unknown as six3 eye targets, are downregulated in the mutant. We show that six3 is required for lens formation, acting primarily in developing retina during neurulation through BMP and Notch signaling, and that mab21l1/mab21l2 regulate(s) this BMP activity. This work reveals previously unrecognized essential roles for six3 in eye development, identifying its key role in signaling needed for lens formation, and acting independently of pax6 activity.
SUMMARY STATEMENT
This study identifies the six3 transcription factor as the mediator of key inductive signals driving lens formation, acting independently of pax6 in early phases of lens formation.
... Mab21l1 null mice are viable but show severe bilateral microphthalmia with a small malformed lens and absence of the iris and ciliary body [PMID 12642482]. Null mice also show delayed calvarial development and male infertility with hypoplasia of the preputial glands [6,7]. Heterozygous mice apparently normal. ...
... This family appear to be phenotypically very similar to those we have identified and this is further support for the causative nature of substitutions affecting MAB21L1 Arg51. It is interesting that the severe eye malformations in Mab21l1 R51L/R51L animals resemble those reported in Mab21l1 null animals [6]. In contradistinction the eye phenotype in null humans is significantly milder than that seen in mice or indeed heterozygous Arg51 substitutions in humans. ...
Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1 . These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2 , a close homolog of MAB21L1 , cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.
... The male abnormal gene family 21 (mab21), was first identified in C. elegans [1]. Since its identification, mab21L1 gene was found to control development of both eye and brain [2][3][4][5][6][7][8][9]. Moreover, it also has a role in regulating axis and dorsal-ventral patterning as well as heart and liver development [3,4,10]. ...
... Later it was found that Mab21L1 is a lens lineage-specific transcription factor [100]. It has an important role in regulating lens development [5,[7][8][9]11]. Mutations in human Mab21L1 gene causes aberrations in lens ectoderm morphogenesis and lead to congenital cerebellar, ocular, craniofacial and genital (COFG) syndrome [101,102]. ...
... The ocular abnormalities include microphthalmia, coloboma and cataracts [7,9]. Similar to human mab21L1 gene mutations, Mab21L1-deficient mice display severe cellautonomous defects in lens placode invagination due to impaired cell proliferation and survival [5] and other deficiency [6,101]. Using mouse lens epithelial cells, αTN4-1 and the stable lines expressing P3X-Flag-CMV-10 vector, or human mab21L1 gene cDNA, here we demonstrated that human mab21L1 gene cDNAexpression lens epithelial cells are much resistant against stress condition of okadaic acid treatment than the vectortransfected cells (Figure 2). ...
The male abnormal gene family 21 (mab21), was initially identified in C. elegans. Since its identification, studies from different groups have shown that it regulates development of ocular tissues, brain, heart and liver. However, its functional mechanism remains largely unknown. Here, we demonstrate that Mab21L1 promotes survival of lens epithelial cells. Mechanistically, Mab21L1 upregulates expression of αB-crystallin. Moreover, our results show that αB-crystallin prevents stress-induced phosphorylation of p53 at S-20 and S-37 through abrogating the activation of the upstream kinases, ATR and CHK1. As a result of suppressing p53 activity by αB-crystallin, Mab21L1 downregulates expression of Bak but upregulates Mcl-1 during stress insult. Taken together, our results demonstrate that Mab21L1 promotes survival of lens epithelial cells through upregulation of αB-crystallin to suppress ATR/CHK1/p53 pathway.
... The final cluster annotation was assigned to the cell type hypothesis left standing after this process. The specificity of the gene expression sets that lead to the assignment with respect to the other clusters was evaluated globally by plotting a heatmap displaying all the marker genes by cluster, cell type, or batch (Bosze et al., 2020;Cajal et al, 2012;Chen et al, 2017;Cohen-Salmon et al, 1997, Crespo-Enriquez et al, 2012Gitton et al, 2011;Kasberg et al, 2013;Kumamoto and Hanashima et al, 2017;Kwon et al, 2010;Lu et al., 2019;McLarren et al., 2003;Pan and Thomson, 2007;Qu et al, 2008;Seo et al, 2017;Soldatov et al, 2019;Tahayato et al, 2003;Yamada et al, 2003). ...
... Both correlation coefficients were then averaged to obtain the top genes anticorrelated to both signatures and therefore most indicative of a cell progenitor status. Some of these genes possessed known links to anterior placode and epithelium development (Imuta et al, 2009;Yamada et al, 2003;Zhou et al, 2010). The computed correlation coefficients are available in Table S4. ...
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1⁺ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.
... In FoxE3 and Pitx3 mutant mice, the lens vesicle forms but fails to separate from the surface ectoderm, leading to disorganized rudimentary lenses (Ho et al., 2009). Notably, Mab21l1-null mice exhibit severe cellautonomous defects in lens placode invagination due to impaired cell proliferation and survival (Yamada et al., 2003); this provided a unique model with which to study early lens placode formation. ...
... In humans, MAB21L2 mutations cause similar defects, with the addition of eye malformations and skeletal dysplasia (Rainger et al., 2014). Mab21l1-deficient mice can survive and grow to adult developmental stages (Yamada et al., 2003); however, they display severe microphthalmia and mild atrophy of the preputial glands with the same abdominal ectoderm origin as the human scrotum (Yamada et al., 2003). Similar to Mab21l1-deficient mouse phenotypes, human MAB21L1 mutations cause various ocular abnormalities, such as microphthalmia, coloboma and/or cataracts (Seese et al., 2021), in addition to facial dysmorphisms, cerebellar hypoplasia and scrotal agenesis; this disease is called cerebellar, ocular, craniofacial and genital (COFG) syndrome (Rad et al., 2019). ...
... In humans, MAB21L2 mutations cause similar defects, with the addition of eye malformations and skeletal dysplasia (Rainger et al., 2014). Mab21l1-deficient mice can survive and grow to adult developmental stages (Yamada et al., 2003); however, they display severe microphthalmia and mild atrophy of the preputial glands with the same abdominal ectoderm origin as the human scrotum (Yamada et al., 2003). Similar to Mab21l1-deficient mouse phenotypes, human MAB21L1 mutations cause various ocular abnormalities, such as microphthalmia, coloboma and/or cataracts (Seese et al., 2021), in addition to facial dysmorphisms, cerebellar hypoplasia and scrotal agenesis; this disease is called cerebellar, ocular, craniofacial and genital (COFG) syndrome (Rad et al., 2019). ...
Mutations in human MAB21L1 cause aberrations in lens ectoderm morphogenesis and lead to congenital cerebellar, ocular, craniofacial, and genital (COFG) syndrome. Murine Mab21l1-null mutations cause severe cell-autonomous defects in lens formation leading to microphthalmia, and therefore is used as a mouse model for COFG syndrome. In this study, we investigated the early-onset, single-cell-level phenotypes of murine Mab21l1-null lens ectoderms using electron microscopy (EM) and single-cell RNA sequencing (scRNA-seq). EM and immunohistochemical analyses indicated endoplasmic reticulum stress in the 24- to 26-somite stage in Mab21l1-null lens placodes. scRNA-seq analysis revealed that 131 genes were downregulated and 148 were upregulated in Mab21l1-null lens ectoderms relative to the wild type. We successfully identified 21 lens-specific genes that were downregulated in Mab21l1-null cells including three key genes involved in lens formation: Pitx3, Maf, and Sfrp2. Moreover, gene ontology analysis of the 279 differentially expressed genes indicated enrichment in housekeeping genes associated with DNA/nucleotide metabolism prior to cell death. These findings suggest that MAB21L1 acts as a nuclear factor that modulates not only lens-specific gene expression but also DNA/nucleotide metabolic processes during lens placode formation.
