Genesis [JOURNAL]
Sun
200 papers
Function of chromatin modifier Hmgn1 during neural crest and craniofacial development.
Ihewulezi C, Saint-Jeannet JP
Genesis · 2021 Oct · PMID 34478234 · Full text
The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a networ...
The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context.
Cloning, expression, and characteristic analysis of the novel β-galactosidase from silkworm, Bombyx mori.
Yi Y, Li J, Zong Z … +6 more , Liu X, Song H, Wang H, Zhang Z, Zhang H, Li Y
Genesis · 2021 Sep · PMID 34449115 · Publisher ↗
β-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the β-galactosidase gene of silkworm (BmGal), whose cDNA compri...
β-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the β-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect β-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal-spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.
The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2.
Howard LJ, Reichert MC, Evans TA
Genesis · 2021 Sep · PMID 34411419 · Full text
Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 doma...
Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N-terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. While Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles depend on its Slit-binding Ig1 domain, we used a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to replace the endogenous robo2 gene with a robo2 variant lacking the Ig1 domain (robo2∆Ig1). We compare the expression and localization of Robo2∆Ig1 protein with full-length Robo2 in embryonic neurons in vivo and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that the removal of the Ig1 domain from Robo2∆Ig1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2∆Ig1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.
Targeted deletion of Atoh8 results in severe hearing loss in mice.
Tang Q, Xie MY, Zhang YL … +3 more , Xue RY, Zhu XH, Yang H
Genesis · 2021 Sep · PMID 34402594 · Full text
Atoh8, also named Math6, is a bHLH gene reported to have important functions in the developing nervous system, pancreas and kidney. However, the expression pattern and function of Atoh8 in the inner ear are still unclear...
Atoh8, also named Math6, is a bHLH gene reported to have important functions in the developing nervous system, pancreas and kidney. However, the expression pattern and function of Atoh8 in the inner ear are still unclear. To study the function of Atoh8 in the developing mouse inner ear, we performed targeted deletion of Atoh8 by intercrossing Atoh8 mice. We studied the expression pattern of Atoh8 in the inner ear and found interesting results that Atoh8-null (Atoh8 ) mice were viable but smaller than their littermates and they were severely hearing impaired, which was confirmed by hearing tests (ABR, DPOAE). We collected 129 viable newborns from 18 litters by crossing Atoh8 mice and found that the distributions of Atoh8 , Atoh8 and wild type were very close to their expected Mendelian ratio by χ testing. However, no remarkable morphological changes in cochleae in mutant mice were detected under plastic sectioning and electron microscopy. No remarkable differences in the expression of Myosin6, Prestin, TrkC, GAD65, Tuj1, or Calretinin were detected between the mutant mice and the control mice. These findings indicate that Atoh8 plays an important role in the development of normal hearing, while further studies are required to elucidate its exact function in hearing.
Sox9CreER-mediated deletion of β-catenin in palatal mesenchyme results in delayed palatal elevation accompanied with repressed canonical Wnt signaling and reduced actin polymerization.
Pang X, Wang X, Wang Y … +5 more , Pu L, Shi J, Burdekin N, Shi B, Li C
Genesis · 2021 Sep · PMID 34390177 · Publisher ↗
Cleft palate is a good model to pushing us toward a deeper understanding of the molecular mechanisms of spatiotemporal patterns in tissues and organisms because of the multiple-step processes such as elevation and fusion...
Cleft palate is a good model to pushing us toward a deeper understanding of the molecular mechanisms of spatiotemporal patterns in tissues and organisms because of the multiple-step processes such as elevation and fusion. Previous studies have shown that the epithelial β-catenin is crucial for palatal fusion, however, the function of the mesenchymal β-catenin remains elusive. We investigate the role of mesenchymal β-catenin in palatal development by generating a β-catenin conditional knockout mouse (CKO) (Sox9CreER; Ctnnb1 ). We found that the CKO mice exhibited delayed palatal elevation, leading to cleft palate in both in vivo and ex vivo. Abnormal cell proliferation and repressed mesenchymal canonical Wnt signaling were found in the CKO palate. Interestingly, Filamentous actin (F-actin) polymerization was significantly reduced in the palatal mesenchyme of mutant embryos. Furthermore, overexpression of adenovirus-mediated transfection with Acta1 in the mutant could help to elevate the palatal shelves but could not prevent cleft palate in ex vivo. Our results suggest that conditionally knock out β-catenin in the palatal mesenchyme by Sox9CreER leading to delayed palatal elevation, which results in repressed mesenchymal canonical Wnt signaling, decreased cell proliferation, and reduced actin polymerization, finally causes cleft palate.
An inducible Cldn11-CreER mouse line for selective targeting of lymphatic valves.
Ortsäter H, Hernández-Vásquez MN, Ulvmar MH … +2 more , Gow A, Mäkinen T
Genesis · 2021 Aug · PMID 34338433 · Publisher ↗
Luminal valves of collecting lymphatic vessels are critical for maintaining unidirectional flow of lymph and their dysfunction underlies several forms of primary lymphedema. Here, we report on the generation of a transge...
Luminal valves of collecting lymphatic vessels are critical for maintaining unidirectional flow of lymph and their dysfunction underlies several forms of primary lymphedema. Here, we report on the generation of a transgenic mouse expressing the tamoxifen inducible CreER under the control of Cldn11 promoter that allows, for the first time, selective and temporally controlled targeting of lymphatic valve endothelial cells. We show that within the vasculature CLDN11 is specifically expressed in lymphatic valves but is not required for their development as mice with a global loss of Cldn11 display normal valves in the mesentery. Tamoxifen treated Cldn11-CreER mice also carrying a fluorescent Cre-reporter displayed reporter protein expression selectively in lymphatic valves and, to a lower degree, in venous valves. Analysis of developing vasculature further showed that Cldn11-CreER -mediated recombination is induced during valve leaflet formation, and efficient labeling of valve endothelial cells was observed in mature valves. The Cldn11-CreER mouse thus provides a valuable tool for functional studies of valves.
An N-terminal fusion allele to study melanin concentrating hormone receptor 1.
Jasso KR, Kamba TK, Zimmerman AD … +8 more , Bansal R, Engle SE, Everett T, Wu CH, Kulaga H, Reed RR, Berbari NF, McIntyre JC
Genesis · 2021 Aug · PMID 34124835 · Full text
Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of...
Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein ( MCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.
Generation and characterization of a tamoxifen-inducible Vsx1-CreER line to target V2 interneurons in the mouse developing spinal cord.
Baudouin C, Pelosi B, Courtoy GE … +2 more , Achouri Y, Clotman F
Genesis · 2021 Aug · PMID 34080769 · Publisher ↗
In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains...
In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreER BAC transgenic mouse line that drives CreER recombinase expression mimicking endogenous Vsx1 expression pattern in the developing spinal cord. We showed that the Vsx1-CreER transgene can mediate recombination in V2 precursors with a high efficacy and specificity. Lineage tracing demonstrated that all the V2 interneurons in the mouse developing spinal cord derive from cells expressing Vsx1. Finally, we confirmed that V2 precursors generate additional V2 populations that are not characterized yet. Thus, the Vsx1-CreER line described here is a useful genetic tool for lineage tracing and for functional studies of the mouse spinal V2 interneurons.
Setd5 is required in cardiopharyngeal mesoderm for heart development and its haploinsufficiency is associated with outflow tract defects in mouse.
Cheung MY, Roberts C, Scambler P … +1 more , Stathopoulou A
Genesis · 2021 Aug · PMID 34050709 · Full text
Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at...
Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at the gene or protein level. In this article we took advantage of an online dataset of high throughput screening of mutations that are embryonic lethal in mice. Our aim was to identify new genes where the loss of function caused cardiovascular phenotypes resembling the 22q11.2 deletion syndrome models, that is, heterozygous and homozygous loss of Tbx1. One gene with a potentially haploinsufficient phenotype was identified, Setd5, thought to be involved in chromatin modification. We found murine Setd5 haploinsufficiency to be associated with double outlet right ventricle and perimembranous ventricular septal defect, although no genetic interaction with Tbx1 was detected. Conditional mutagenesis revealed that Setd5 was required in cardiopharyngeal mesoderm for progression of the heart tube through the ballooning stage to create a four-chambered heart.
The mechanism and function of super enhancer RNA.
Xiao S, Huang Q, Ren H … +1 more , Yang M
Genesis · 2021 Jun · PMID 34028961 · Publisher ↗
Super enhancer (SE) is a cluster of enhancers that has a stronger ability to promote transcription compared to the typical enhancer (TE). Similar to TE, which can transcribe enhancer RNA (eRNA), SE produces super enhance...
Super enhancer (SE) is a cluster of enhancers that has a stronger ability to promote transcription compared to the typical enhancer (TE). Similar to TE, which can transcribe enhancer RNA (eRNA), SE produces super enhancer RNA (seRNA). The activation of SE is cell and tissue-specific, and the SE-associated genes are mostly linked to the cell fate. This is demonstrated by the important role-played by SE in the embryonic stem cell (ESC) and multiple cancer development. SeRNA regulates transcription in both cis and trans configuration, and those located in the cytoplasm mediates various cell activities. However, the functions of seRNAs are unclear, and most of them have a synergistic effect with SE and SE-associated genes. In this mini review, we summarized the mechanisms of seRNA and functions of both SE and seRNA.
Constitutive expression of spliced XBP1 causes perinatal lethality in mice.
Xu Q, Zhang H, Wang S … +2 more , Qin C, Lu Y
Genesis · 2021 Jun · PMID 33891366 · Full text
Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA tha...
Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor. XBP1S is a key mediator of the IRE1 branch that is essential for alleviating ER stress. We generated a novel mouse strain (referred to as "Xbp1 " mice) that constitutively expressed XBP1S after Cre recombinase-mediated recombination. Further breeding of these mice with Twist2 Cre recombinase (Twist2-Cre) knock-in mice generated Twist2-Cre;Xbp1 mice. Most Twist2-Cre;Xbp1 mice died shortly after birth. Reverse-transcription polymerase chain reaction (RT-PCR) showed that constitutive expression of XBP1S occurred in various mouse tissues examined, but not in the brain. Immunohistochemistry confirmed that although the immunostaining signals for total XBP1 (XBP1U and XBP1S) were found in the calvarial bones in both Twist2-Cre;Xbp1 and control mice, the signals for XBP1S were only detected in the Twist2-Cre;Xbp1 mice, but not in the control mice. These results suggest that a precise control of XBP1S production is essential for normal mouse development.
Generation of a new mouse line with conditionally activated signaling through the BMP receptor, ACVR1: A tool to characterize pleiotropic roles of BMP functions.
Yang J, Toda Nakamura M, Hallett SA … +6 more , Ueharu H, Zhang H, Kelley K, Fukuda T, Komatsu Y, Mishina Y
Genesis · 2021 Jun · PMID 33851764 · Full text
BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei in...
BMP signaling plays pleiotropic roles in various tissues during embryogenesis and after birth. We have previously generated a constitutively activated Acvr1(ca-Acvr1) transgenic mouse line (line L35) through pronuclei injection to investigate impacts of enhanced BMP signaling in a tissue specific manner. However, line L35 shows a restricted expression pattern of the transgene. Here, we generated another ca-Acvr1 transgenic line, line A11, using embryonic stem (ES) transgenesis. The generated line A11 shows distinctive phenotypes from line L35, along with very limited expression levels of the transgene. When the transgene is activated in the neural crest cells in a Cre-dependent manner, line A11 exhibits cleft palate and shorter jaws, while line L35 develops ectopic cartilages and highly hypomorphic facial structures. When activated in limb buds, line A11 develops organized but smaller limb skeletal structures, while line L35 forms disorganized limbs with little mineralization. Additionally, no heterotopic ossification (HO) is identified in line A11 when bred with NFATc1-Cre mice even after induction of tissue injury, which is an established protocol for HO for line L35. Therefore, the newly generated conditional ca-Acvr1 mouse line A11 provides an additional resource to dissect highly context dependent functions of BMP signaling in development and disease.
Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.
Onjiko RM, Nemes P, Moody SA
Genesis · 2021 Jun · PMID 33826226 · Full text
The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in th...
The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.
The adhesion GPCR Adgrg6 (Gpr126): Insights from the zebrafish model.
Baxendale S, Asad A, Shahidan NO … +2 more , Wiggin GR, Whitfield TT
Genesis · 2021 Apr · PMID 33735533 · Full text
Adhesion GPCRs are important regulators of conserved developmental processes and represent an untapped pool of potential targets for drug discovery. The adhesion GPCR Adgrg6 (Gpr126) has critical developmental roles in S...
Adhesion GPCRs are important regulators of conserved developmental processes and represent an untapped pool of potential targets for drug discovery. The adhesion GPCR Adgrg6 (Gpr126) has critical developmental roles in Schwann cell maturation and inner ear morphogenesis in the zebrafish embryo. Mutations in the human ADGRG6 gene can result in severe deficits in peripheral myelination, and variants have been associated with many other disease conditions. Here, we review work on the zebrafish Adgrg6 signaling pathway and its potential as a disease model. Recent advances have been made in the analysis of the structure of the Adgrg6 receptor, demonstrating alternative structural conformations and the presence of a conserved calcium-binding site within the CUB domain of the extracellular region that is critical for receptor function. Homozygous zebrafish adgrg6 hypomorphic mutants have been used successfully as a whole-animal screening platform, identifying candidate molecules that can influence signaling activity and rescue mutant phenotypes. These compounds offer promise for further development as small molecule modulators of Adgrg6 pathway activity.
VPS4B deficiency causes early embryonic lethality and induces signal transduction disorders of cell endocytosis.
Chen D, He F, Lu T … +6 more , Huang J, Li M, Cai D, Huang C, Chen D, Xiong F
Genesis · 2021 Apr · PMID 33682352 · Publisher ↗
VPS4B (vacuolar protein sorting 4B), a member of the ATPase associated with diverse cellular activities (AAA) protein family, is a component of the endosomal sorting complexes required for transport machinery which regul...
VPS4B (vacuolar protein sorting 4B), a member of the ATPase associated with diverse cellular activities (AAA) protein family, is a component of the endosomal sorting complexes required for transport machinery which regulates the internalization and lysosomal degradation of membrane proteins. We previously reported that VPS4B is one of the pathogenic genes related to dentin dysplasia type I, although its function was largely unknown. To investigate the role of VPS4B in tooth development, we deleted the Vps4b gene in mice. We found that heterozygous knockout mice (Vps4b ) developed normally and were fertile. However, homozygous deletion of the Vps4b gene resulted in early embryonic lethality of Vps4b mice at approximately embryonic day 9.5 (E9.5). To investigate the underlying molecular mechanisms, we examined the molecular functions of VPS4B in vivo and in vitro. Cell experiments showed that VPS4B influenced the proliferation, apoptosis, and cell cycle of transfected human neuroblastoma cells (IMR-32 cells) with over-expression or knockdown of VPS4B. Moreover, qRT-PCR detection showed that the mRNA expression levels of apoptosis-, cell cycle-, and endocytosis-related genes was significantly down or up-regulated in RNA interference-mediated knockdown of VPS4B in IMR-32 cells and Vps4b E12.5 embryos. We accordingly speculated that signal transduction disorders of cell endocytosis are a contributing factor to the prenatal lethality of Vps4b mice.
Tmem100-BAC-EGFP mice to selectively mark and purify embryonic endothelial cells of large caliber arteries in mid-gestational vascular formation.
Kinugasa-Katayama Y, Watanabe Y, Hisamitsu T … +9 more , Arima Y, Liu NM, Tomimatsu A, Harada Y, Arai Y, Urasaki A, Kawamura T, Saito Y, Nakagawa O
Genesis · 2021 Apr · PMID 33651473 · Publisher ↗
Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell...
Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.
Xenopus to the rescue: A model to validate and characterize candidate ciliopathy genes.
Rao VG, Kulkarni SS
Genesis · 2021 Feb · PMID 33576572 · Publisher ↗
Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting mill...
Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting millions of lives worldwide. Yet, our understanding of the gene regulatory networks that control cilia assembly and functions remain incomplete. With the advances in next-generation sequencing technologies, we can now rapidly predict pathogenic variants from hundreds of ciliopathy patients. While the pace of candidate gene discovery is exciting, most of these genes have never been previously implicated in cilia assembly or function. This makes assigning the disease causality difficult. This review discusses how Xenopus, a genetically tractable and high-throughput vertebrate model, has played a central role in identifying, validating, and characterizing candidate ciliopathy genes. The review is focused on multiciliated cells (MCCs) and diseases associated with MCC dysfunction. MCCs harbor multiple motile cilia on their apical surface to generate extracellular fluid flow inside the airway, the brain ventricles, and the oviduct. In Xenopus, these cells are external and present on the embryonic epidermal epithelia, facilitating candidate genes analysis in MCC development in vivo. The ability to introduce patient variants to study their effects on disease progression makes Xenopus a powerful model to improve our understanding of the underlying disease mechanisms and explain the patient phenotype.
Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems.
Adil MT, Henry JJ
Genesis · 2021 Feb · PMID 33576188 · Publisher ↗
Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, hom...
Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.
Atypical protein kinase C is essential for embryonic vascular development in mice.
Chen Z, Duan Y, Wang H … +6 more , Tang H, Wang S, Wang X, Liu J, Fang X, Ouyang K
Genesis · 2021 Mar · PMID 33547760 · Full text
The atypical PKC (aPKC) subfamily constitutes PKCζ and PKCλ in mice, and both aPKC isoforms have been proposed to be involved in regulating various endothelial cell (EC) functions. However, the physiological function of...
The atypical PKC (aPKC) subfamily constitutes PKCζ and PKCλ in mice, and both aPKC isoforms have been proposed to be involved in regulating various endothelial cell (EC) functions. However, the physiological function of aPKC in ECs during embryonic development has not been well understood. To address this question, we utilized Tie2-Cre to delete PKCλ alone (PKCλ-SKO) or both PKCλ and PKCζ (DKO) in ECs, and found that all DKO mice died at around the embryonic day 11.5 (E11.5), whereas a small proportion of PKCλ-SKO mice survived till birth. PKCλ-SKO embryos also exhibited less phenotypic severity than DKO embryos at E10.5 and E11.5, suggesting a potential compensatory role of PKCζ for PKCλ in embryonic ECs. We then focused on DKO embryos and investigated the effects of aPKC deficiency on embryonic vascular development. At E9.5, deletion of both aPKC isoforms reduced the diameters of vitelline artery and vein, and decreased branching from both vitelline vessels in yolk sac. Ablation of both aPKC isoforms also disrupted embryonic angiogenesis in head and trunk at the same stage, increasing apoptosis of both ECs and non-ECs. Taken together, our results demonstrated that aPKC in ECs plays an essential role in regulating cell apoptosis, angiogenesis, and embryonic survival.
Aquatic models of human ciliary diseases.
Corkins ME, Krneta-Stankic V, Kloc M … +1 more , Miller RK
Genesis · 2021 Feb · PMID 33496382 · Full text
Cilia are microtubule-based structures that either transmit information into the cell or move fluid outside of the cell. There are many human diseases that arise from malfunctioning cilia. Although mammalian models provi...
Cilia are microtubule-based structures that either transmit information into the cell or move fluid outside of the cell. There are many human diseases that arise from malfunctioning cilia. Although mammalian models provide vital insights into the underlying pathology of these diseases, aquatic organisms such as Xenopus and zebrafish provide valuable tools to help screen and dissect out the underlying causes of these diseases. In this review we focus on recent studies that identify or describe different types of human ciliopathies and outline how aquatic organisms have aided our understanding of these diseases.
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