Generation of a new six1-null line in Xenopus tropicalis for study of development and congenital disease

The vertebrate Six (Sine oculis homeobox) family of homeodomain transcription factors play critical roles in the development of several organs. Six1 plays a central role in cranial placode development, including the precursor tissues of the inner ear, as well as other cranial sensory organs and the kidney. In humans, mutations in SIX1 underlie some cases of branchio-oto-renal syndrome (BOR), which is characterized by moderate to severe hearing loss. We utilized CRISPR/Cas9 technology to establish a six1 mutant line in Xenopus tropicalis that is available to the research community. We demonstrate that at larval stages, the six1-null animals show severe disruptions in gene expression of putative Six1 target genes in the otic vesicle, cranial ganglia, branchial arch and neural tube. At tadpole stages, six1-null animals display dysmorphic Meckel's, ceratohyal and otic capsule cartilage morphology. This mutant line will be of value for the study of the development of several organs as well as congenital syndromes that involve these tissues.

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Generation of a new six1 ‐null line in Xenopus tropicalis for study of development and congenital disease

genesis ◽

10.1002/dvg.23453 ◽

2021 ◽

Author(s):

Kelsey Coppenrath ◽

Andre L. P. Tavares ◽

Nikko‐Ideen Shaidani ◽

Marcin Wlizla ◽

Sally A. Moody ◽

...

Keyword(s):

Xenopus Tropicalis ◽

Null Line ◽

Congenital Disease

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Meis2 Is Required for Inner Ear Formation and Proper Morphogenesis of the Cochlea

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.679325 ◽

2021 ◽

Vol 9 ◽

Author(s):

María Beatriz Durán Alonso ◽

Victor Vendrell ◽

Iris López-Hernández ◽

María Teresa Alonso ◽

Donna M. Martin ◽

...

Keyword(s):

Nervous System ◽

Inner Ear ◽

Cell Line ◽

Peripheral Nervous System ◽

Target Genes ◽

Otic Vesicle ◽

Vesicle Formation ◽

Tissue Specific ◽

Cochlear Duct ◽

Candidate Target

Meis genes have been shown to control essential processes during development of the central and peripheral nervous system. Here we have explored the roles of the Meis2 gene during vertebrate inner ear induction and the formation of the cochlea. Meis2 is expressed in several tissues required for inner ear induction and in non-sensory tissue of the cochlear duct. Global inactivation of Meis2 in the mouse leads to a severely reduced size of the otic vesicle. Tissue-specific knock outs of Meis2 reveal that its expression in the hindbrain is essential for otic vesicle formation. Inactivation of Meis2 in the inner ear itself leads to an aberrant coiling of the cochlear duct. By analyzing transcriptomes obtained from Meis2 mutants and ChIPseq analysis of an otic cell line, we define candidate target genes for Meis2 which may be directly or indirectly involved in cochlear morphogenesis. Taken together, these data show that Meis2 is essential for inner ear formation and provide an entry point to unveil the network underlying proper coiling of the cochlear duct.

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Gsx2 but not Gsx1 is necessary for early forebrain patterning and long-term survival in zebrafish

10.1101/2021.09.13.460150 ◽

2021 ◽

Cited By ~ 1

Author(s):

RA Coltogirone ◽

EI Sherfinski ◽

ZA Dobler ◽

SN Peterson ◽

AR Andlinger ◽

...

Keyword(s):

Transcription Factors ◽

Gene Networks ◽

Target Genes ◽

Molecular Genetic ◽

Brain Regions ◽

Transcription Activator ◽

Term Survival ◽

Altered Expression ◽

Larval Stages

ABSTRACTCentral nervous system (CNS) development is regulated by regionally expressed transcription factors that impart initial cell identity, connectivity, and function to neural circuits through complex molecular genetic cascades. genomic screen homeobox 1 and 2 (gsx1 and gsx2) encode homeobox transcription factors expressed in the developing CNS in multiple vertebrates examined to date. However, we have limited knowledge of the expression of these transcription factors and the gene networks that they regulate across developing brain regions in zebrafish. The objective of this study was to comprehensively examine gsx1 and gsx2 expression throughout neurodevelopment and characterize gsx1 and gsx2 mutants to study the essential roles of these closely related transcription factors. Using RT-PCR, whole-mount in situ hybridization (WISH), and fluorescence in situ hybridization, we examine gsx1 and gsx2 expression from early embryonic to late larval stages. gsx1 is expressed initially in the hindbrain and diencephalon and later in the optic tectum, pretectum, and cerebellar plate. Comparatively, gsx2 is expressed in the early telencephalon and later in the pallium and olfactory bulb. gsx1 and gsx2 are regionally co-expressed in the hypothalamus, preoptic area, and hindbrain, however rarely co-localize in the same cells. To identify forebrain target genes, we utilize mutants made with Transcription activator-like effector nucleases (TALEN). gsx1 mutant zebrafish exhibit stunted growth, however, they survive through adulthood and are fertile. gsx2 mutant zebrafish experience swim bladder inflation failure that prevents survival past larval stage. Using WISH and RT-qPCR we demonstrate altered expression of genes including, distal-less homeobox genes and forkhead box gene foxp2. This work provides novel tools with which other target genes and functions of Gsx1 and Gsx2 can be characterized across the CNS to better understand the unique and overlapping roles of these highly conserved transcription factors.

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Expression of the mouse goosecoid gene during mid-embryogenesis may mark mesenchymal cell lineages in the developing head, limbs and body wall

Development ◽

10.1242/dev.117.2.769 ◽

1993 ◽

Vol 117 (2) ◽

pp. 769-778 ◽

Cited By ~ 22

Author(s):

S.J. Gaunt ◽

M. Blum ◽

E.M. De Robertis

Keyword(s):

Body Wall ◽

Homeobox Gene ◽

Mesenchymal Cell ◽

Primitive Streak ◽

Branchial Arch ◽

Sensory Epithelium ◽

The Body ◽

Otic Vesicle ◽

Mouse Development ◽

Branchial Cleft

After an earlier, transient phase of expression in the developing primitive streak of 6.4- to 6.8-day mouse embryos, the homeobox gene goosecoid is now shown to be expressed in a later phase of mouse development, from 10.5 days onwards. The later, spatially restricted domains of goosecoid expression are detected in the head, limbs and ventrolateral body wall. At all sites, the domains of expression are first detected in undifferentiated tissue, and then expression persists as these tissues undergo subsequent morphogenesis. For example, goosecoid expression is noted in the first branchial arch at 10.5 days, and then expression persists as this tissue undergoes morphogenesis to form the lower jaw and the body of the tongue. Expression in tissues around the first branchial cleft persists as these undergo morphogenesis to form the base of the auditory meatus and eustachian tube. Expression in tissues around the newly formed nasal pits persists as these elongate to form the nasal chambers. Expression in the ventral epithelial lining of the otic vesicle persists as this eventually gives rise to the non-sensory epithelium of the cochlea. Expression in the proximal limb buds and ventrolateral body wall persists as these tissues undergo morphogenesis to form proximal limb structures and ventral ribs respectively. Our findings lead us to suggest that the goosecoid gene product plays a role in spatial programming within discrete embryonic fields, and possibly lineage compartments, during organogenesis stages of mouse development.

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Developmental role of plk4 in Xenopus laevis and Danio rerio: implications for Seckel Syndrome

Biochemistry and Cell Biology ◽

10.1139/bcb-2015-0003 ◽

2015 ◽

Vol 93 (4) ◽

pp. 396-404 ◽

Cited By ~ 1

Author(s):

Candace Elaine Rapchak ◽

Neeraj Patel ◽

John Hudson ◽

Michael Crawford

Keyword(s):

Cell Polarity ◽

Planar Cell Polarity ◽

Expression Patterns ◽

Branchial Arch ◽

Multiple Roles ◽

Otic Vesicle ◽

Seckel Syndrome ◽

Brain Expression

The polo-like kinases are a family of conserved serine/threonine kinases that play multiple roles in regulation of the cell cycle. Unlike its four other family members, the role of Plk4 in embryonic development has not been well characterized. In mice, Plk4−/− embryos arrest at E7.5, just prior to the initiation of somitogenesis. This has led to the hypothesis that Plk4 expression may be essential to somitogenesis. Recently characterized human mutations lead to Seckel Syndrome. Riboprobe in situ hybridization revealed that plk4 is ubiquitously expressed during early stages of development of Xenopus and Danio; in later stages, expression in frogs restricts to somites as well as eye, otic vesicle, and branchial arch, and brain. Expression patterns in fish remain ubiquitous. Both somite and eye development require planar cell polarity, and disruption of plk4 function in frog by means of morpholino-mediated translational knockdown yields orientational disorganization of both these structures. These results provide the first steps in defining a new role for plk4 in organogenesis and implies a role in planar cell polarity, segmentation, and in recently described PLK4 mutations in human.

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MiR-92 Family Members Form a Cluster Required for Notochord Tubulogenesis in Urochordate Ciona savignyi

Genes ◽

10.3390/genes12030406 ◽

2021 ◽

Vol 12 (3) ◽

pp. 406

Author(s):

Libo Yang ◽

Xiaoming Zhang ◽

Chengzhang Liu ◽

Jin Zhang ◽

Bo Dong

Keyword(s):

Signaling Pathways ◽

Planar Cell Polarity ◽

Target Genes ◽

Expression Patterns ◽

Family Members ◽

Mitogen Activated Protein Kinase ◽

Mirna Cluster ◽

Ciona Savignyi ◽

Larval Stages ◽

Regulatory Processes

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18–31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster’s function during evolution.

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Mutations in SIX1 associated with Branchio-oto-renal Syndrome (BOR) differentially affect otic expression of putative target genes

10.1101/2021.06.11.447956 ◽

2021 ◽

Author(s):

Tanya Mehdizadeh ◽

Himani Datta Majumdar ◽

Sahra Ahsan ◽

Andre Luiz Pasqua Tavares ◽

Sally A Moody

Keyword(s):

Gene Expression ◽

Target Genes ◽

Otic Vesicle ◽

Wild Type ◽

Single Nucleotide ◽

Putative Target ◽

Individual Effects ◽

Factor Interactions ◽

Ability To Drive

Single nucleotide mutations in SIX1 are causative in some individuals diagnosed with branchiootic/branchio-oto-renal (BOR) syndrome. To test whether these mutations have differential effects on otic gene expression, we engineered four BOR mutations in Xenopus six1 and targeted mutant protein expression to the neural crest and cranial placode precursor cells in wild-type embryos. Changes in the otic expression of putative Six1 targets and/or co-factors were monitored by qRT-PCR and in situ hybridization. We found that each mutant had a different combination of effects. The V17E mutant reduced eya2, tspan13, zbtb16 and pa2g4 otic vesicle expression at a frequency indistinguishable from wildtype Six1, but reduced prdm1 more and spry1 less compared to wild-type Six1. For most of these genes, the R110W, W122R and Y129C mutants were significantly less repressive compared to wild-type Six1. Their individual effects varied according to the level at which they were expressed. The R110W, W122R and Y129C mutants also often expanded prdm1 otic expression. Since previous studies showed that all four mutants are transcriptionally deficient and differ in their ability to interact with co-factors such as Eya1, we propose that altered co-factor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression.

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The role of Niemann-Pick type C2 in zebrafish embryonic development

Development ◽

10.1242/dev.194258 ◽

2021 ◽

pp. dev.194258

Author(s):

Wei-Chia Tseng ◽

Ana J Johnson Escauriza ◽

Chon-Hwa Tsai-Morris ◽

Benjamin Feldman ◽

Ryan K. Dale ◽

...

Keyword(s):

Embryonic Development ◽

Notch Signaling Pathway ◽

Otic Vesicle ◽

Unesterified Cholesterol ◽

Lysosomal Disease ◽

Developmental Defects ◽

Larval Stages ◽

Abnormal Head ◽

Niemann Pick

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein which functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, while reducing cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes, no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

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Cross-interactions between two members of the Dlx family of homeobox-containing genes during zebrafish development

Biochemistry and Cell Biology ◽

10.1139/o97-076 ◽

1997 ◽

Vol 75 (5) ◽

pp. 613-622 ◽

Cited By ~ 9

Author(s):

T Zerucha ◽

J -P Muller ◽

N Chartrand ◽

M Ekker

Keyword(s):

Target Genes ◽

Expression Patterns ◽

Homeobox Genes ◽

Otic Vesicle ◽

Zebrafish Embryos ◽

Zebrafish Development ◽

Regulatory Interactions ◽

Dlx Genes ◽

Flanking Region ◽

Multiple Cells

The Dlx homeobox genes of vertebrates are transcribed in multiple cells of the embryo with overlapping patterns but often with different onsets of expression. Here we describe the interaction between two dlx genes, dlx3 and dlx4, during zebrafish development. The observation that dlx3 expression precedes that of dlx4 in the otic vesicle led us to investigate whether dlx3 had the ability to control expression of dlx4. Truncated versions of dlx3 were overexpressed in zebrafish embryos and the expression patterns of dlx4 were examined later in development. Overexpression of truncated forms of Dlx3 or of a Dlx3-Dlx2 chimera was found to result in perturbations in dlx4 expression. In addition, cotransfection experiments indicated the ability of Dlx3 to activate transcription through a 1.7-kb fragment of the 5 prime flanking region of dlx4. These results suggest that dlx4 is one of the target genes of dlx3 in embryos and that cross-regulatory interactions between Dlx genes may be one of the mechanisms responsible for their overlapping expression.

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Histone Demethylase PHF8 Is Required for the Development of the Zebrafish Inner Ear and Posterior Lateral Line

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.566504 ◽

2020 ◽

Vol 8 ◽

Author(s):

Jing He ◽

Zhiwei Zheng ◽

Xianyang Luo ◽

Yongjun Hong ◽

Wenling Su ◽

...

Keyword(s):

Cell Migration ◽

Inner Ear ◽

Lateral Line ◽

Target Genes ◽

Histone Demethylase ◽

Otic Vesicle ◽

Lateral Line System ◽

Potential Candidate ◽

Line System ◽

Posterior Lateral Line

Histone demethylase PHF8 is crucial for multiple developmental processes, and hence, the awareness of its function in developing auditory organs needs to be increased. Using in situ hybridization (ISH) labeling, the mRNA expression of PHF8 in the zebrafish lateral line system and otic vesicle was monitored. The knockdown of PHF8 by morpholino significantly disrupted the development of the posterior lateral line system, which impacted cell migration and decreased the number of lateral line neuromasts. The knockdown of PHF8 also resulted in severe malformation of the semicircular canal and otoliths in terms of size, quantity, and position during the inner ear development. The loss of function of PHF8 also induced a defective differentiation in sensory hair cells in both lateral line neuromasts and the inner ear. ISH analysis of embryos that lacked PHF8 showed alterations in the expression of many target genes of several signaling pathways concerning cell migration and deposition, including the Wnt and FGF pathways. In summary, the current findings established PHF8 as a novel epigenetic element in developing auditory organs, rendering it a potential candidate for hearing loss therapy.

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