Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development.

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

Never-ending story of Brachyury: From short-tailed mice to tailless primates.

The history of developmental biology starts from the almost simultaneous discoveries of the Organizer of axial structures in amphibians by Spemann and Mangold in Freiburg and of the Brachyury mutant in mammals by the Dobrovolskaya-Zavadskaya laboratory at the Curie Institute and its follow-up studies in the Leslie Dunn laboratory at Columbia University. Following the Organizer's discovery, the inductive activity of several other embryonic tissues was found, including that of the ear primordium by Boris Balinsky in Kiev. Initially, the experimental embryological and genetic lines of research existed independently of each other, but after they met at the bench of Salome Gluecksohn, they strengthened and cross-fertilized each other, eventually leading to developmental genetics, which later became known as developmental biology. It appears that the regulatory activities of Brachyury and related T-box proteins in general are at the heart of the development of all vertebrates. These activities are fundamental and have been discovered in several model organisms subjected to mutagenesis, exemplified by the story of George Streisinger's discovery of the no tail mutant in zebrafish. This essay describes the history of Brachyury studies, their connection to an idea of embryonic induction by Organizer, and an impact of Brachyury and related genes on various fields of research from embryology and cell biology to medical genetics and evolutionary theory.

The prolyl isomerase Pin1 stabilizes NeuroD during differentiation of mechanoreceptors.

The peptidyl prolyl cis-trans isomerase Pin1 plays vital roles in diverse cellular processes and pathological conditions. NeuroD is a differentiation and survival factor for a subset of neurons and pancreatic endocrine cells. Although multiple phosphorylation events are known to be crucial for NeuroD function, their mechanisms remain elusive. In this study, we demonstrate that zebrafish embryos deficient in Pin1 displayed phenotypes resembling those associated with NeuroD depletion, characterized by defects in formation of mechanosensory hair cells. Furthermore, zebrafish Pin1 interacts with NeuroD in a phosphorylation-dependent manner. In Pin1-deficient cell lines, NeuroD is rapidly degraded. However, the protein stability of NeuroD is restored upon overexpression of Pin1. These findings suggest that Pin1 functionally regulates NeuroD protein levels by post-phosphorylation cis-trans isomerization during neuronal specification.

Development of the brain ventricular system from a comparative perspective.

The brain ventricular system (BVS) consists of brain ventricles and channels filled with cerebrospinal fluid (CSF). Disturbance of CSF flow has been linked to scoliosis and neurodegenerative diseases, including hydrocephalus. This could be due to defects of CSF production by the choroid plexus or impaired CSF movement over the ependyma dependent on motile cilia. Most vertebrates have horizontal body posture. They retain additional evolutionary innovations assisting CSF flow, such as the Reissner fiber. The causes of hydrocephalus have been studied using animal models including rodents (mice, rats, hamsters) and zebrafish. However, the horizontal body posture reduces the effect of gravity on CSF flow, which limits the use of mammalian models for scoliosis. In contrast, fish swim against the current and experience a forward-to-backward mechanical force akin to that caused by gravity in humans. This explains the increased popularity of the zebrafish model for studies of scoliosis. "Slit-ventricle" syndrome is another side of the spectrum of BVS anomalies. It develops because of insufficient inflation of the BVS. Recent advances in zebrafish functional genetics have revealed genes that could regulate the development of the BVS and CSF circulation. This review will describe the BVS of zebrafish, a typical teleost, and vertebrates in general, in comparative perspective. It will illustrate the usefulness of the zebrafish model for developmental studies of the choroid plexus (CP), CSF flow and the BVS.

Transcriptome profile of the sinoatrial ring reveals conserved and novel genetic programs of the zebrafish pacemaker.

BACKGROUND: Sinoatrial Node (SAN) is part of the cardiac conduction system, which controls the rhythmic contraction of the vertebrate heart. The SAN consists of a specialized pacemaker cell population that has the potential to generate electrical impulses. Although the SAN pacemaker has been extensively studied in mammalian and teleost models, including the zebrafish, their molecular nature remains inadequately comprehended. RESULTS: To characterize the molecular profile of the zebrafish sinoatrial ring (SAR) and elucidate the mechanism of pacemaker function, we utilized the transgenic line sqet33mi59BEt to isolate cells of the SAR of developing zebrafish embryos and profiled their transcriptome. Our analyses identified novel candidate genes and well-known conserved signaling pathways involved in pacemaker development. We show that, compared to the rest of the heart, the zebrafish SAR overexpresses several mammalian SAN pacemaker signature genes, which include hcn4 as well as those encoding calcium- and potassium-gated channels. Moreover, genes encoding components of the BMP and Wnt signaling pathways, as well as members of the Tbx family, which have previously been implicated in pacemaker development, were also overexpressed in the SAR. Among SAR-overexpressed genes, 24 had human homologues implicated in 104 different ClinVar phenotype entries related to various forms of congenital heart diseases, which suggest the relevance of our transcriptomics resource to studying human heart conditions. Finally, functional analyses of three SAR-overexpressed genes, pard6a, prom2, and atp1a1a.2, uncovered their novel role in heart development and physiology. CONCLUSION: Our results established conserved aspects between zebrafish and mammalian pacemaker function and revealed novel factors implicated in maintaining cardiac rhythm. The transcriptome data generated in this study represents a unique and valuable resource for the study of pacemaker function and associated heart diseases.

Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

High Behavioral Variability Mediated by Altered Neuronal Excitability in auts2 Mutant Zebrafish.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. The autism susceptibility candidate 2 (AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. However, the role of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild-type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties. auts2a mutant zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. Using calcium imaging we probed the activity of Mauthner neurons during otic vesicle (OV) stimulation and observed lower probability of activation and reduced calcium transients in the mutants. With direct activation of Mauthner by antidromic stimulation, the threshold for activation in mutants was higher than that in wild-type, even when inhibition was blocked. Taken together, these results point to reduced excitability of Mauthner neurons in auts2a mutant larvae leading to unreliable escape responses. Our results show a novel role for Auts2a in regulating neural excitability and reliability of behavior.

The development of zebrafish pancreas affected by deficiency of Hedgehog signaling.

The pancreas development depends on complex regulation of several signaling pathways, including the Hedgehog (Hh) signaling via a receptor complex component, Smoothened, which deficiency blocks the Hh signaling. Such a defect in birds and mammals results in an annular pancreas. We showed that in developing zebrafish, the mutation of Smoothened or inhibition of Hh signaling by its antagonist cyclopamine caused developmental defects of internal organs, liver, pancreas, and gut. In particular, the pancreatic primordium was duplicated. The two exocrine pancreatic primordia surround the gut. This phenomenon correlates with a significant reduction of the gut's diameter, causing the annular pancreas phenotype.

Evolutionary context can clarify gene names: Teleosts as a case study.

We developed an ex silico evolutionary-based systematic synteny approach to define and name the duplicated genes in vertebrates. The first convention for the naming of genes relied on historical precedent, the order in the human genome, and mutant phenotypes in model systems. However, total-genome duplication that resulted in teleost genomes required the naming of duplicated orthologous genes (ohnologs) in a specific manner. Unfortunately, as we review here, such naming has no defined criteria, and some ohnologs and their orthologs have suffered from incorrect nomenclature, thus creating confusion in comparative genetics and disease modeling. We sought to overcome this barrier by establishing an ex silico evolutionary-based systematic approach to naming ohnologs in teleosts. We developed software and compared gene synteny in zebrafish using the spotted gar genome as a reference, representing the unduplicated ancestral state. Using new criteria, we identified several hundred potentially misnamed ohnologs and validated the principle manually. Also see the video abstract here: https://youtu.be/UKNLa_TvSgY.

Zygotic Genome Activation: Critical Prelude to the Most Important Time of Your Life.

Activation of the embryonic genome during development represents a major developmental transition in all species. The history of its exploration began in the 1950s-1960s, when this idea was put forward and proven experimentally by Alexander Neyfakh. He observed the aberrant development of fish embryos upon X-ray irradiation and noted the different developmental outcomes depending on the stage when fertilized eggs were subjected to irradiation. Neyfakh also discriminated a regional difference of X-irradiation between the nucleus and the cytoplasm. By selecting the X-ray dose causing nuclear damage, he determined the beginning of zygotic transcription, which at that time became known as the morphogenetic function of nuclei. His team defined the link of zygotic transcription with the asynchronization of cell division and cell migration, the two other hallmarks, which along with the morphogenetic function (or the zygotic genome activation), are at the core of the mid-blastula transition during development. Within this framework, current studies using maternal mutants and application of modern methods of whole-embryo and single-cell transcriptomics begin to decipher the molecular mechanisms of the mid-blastula transition (or the maternal-zygotic transition).

An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique.

Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Kcnb1 plays a role in development of the inner ear.

The function of the inner ear depends on the maintenance of high concentrations of K+ ions. The slow-inactivating delayed rectifier Kv2.1/KCNB1 channel works in the inner ear in mammals. The kcnb1 gene is expressed in the otic vesicle of developing zebrafish, suggesting its role in development of the inner ear. In the present study, we found that a Kcnb1 loss-of-function mutation affected development of the inner ear at multiple levels, including otic vesicle expansion, otolith formation, and the proliferation and differentiation of mechanosensory cells. This resulted in defects of kinocilia and stereocilia and abnormal function of the inner ear detected by behavioral assays. The quantitative transcriptional analysis of 75 genes demonstrated that the kcnb1 mutation affected the transcription of genes that are involved in K+ metabolism, cell proliferation, cilia development, and intracellular protein trafficking. These results demonstrate a role for Kv2.1/Kcnb1 channels in development of the inner ear in zebrafish.

Camel regulates development of the brain ventricular system.

Development of the brain ventricular system of vertebrates and the molecular mechanisms involved are not fully understood. The developmental genes expressed in the elements of the brain ventricular system such as the ependyma and circumventricular organs act as molecular determinants of cell adhesion critical for the formation of brain ventricular system. They control brain development and function, including the flow of cerebrospinal fluid. Here, we describe the novel distantly related member of the zebrafish L1-CAM family of genes-camel. Whereas its maternal transcripts distributed uniformly, the zygotic transcripts demonstrate clearly defined expression patterns, in particular in the axial structures: floor plate, hypochord, and roof plate. camel expresses in several other cell lineages with access to the brain ventricular system, including the midbrain roof plate, subcommissural organ, organum vasculosum lamina terminalis, median eminence, paraventricular organ, flexural organ, and inter-rhombomeric boundaries. This expression pattern suggests a role of Camel in neural development. Several isoforms of Camel generated by differential splicing of exons encoding the sixth fibronectin type III domain enhance cell adhesion differentially. The antisense oligomer morpholino-mediated loss-of-function of Camel affects cell adhesion and causes hydrocephalus and scoliosis manifested via the tail curled down phenotype. The subcommissural organ's derivative-the Reissner fiber-participates in the flow of cerebrospinal fluid. The Reissner fiber fails to form upon morpholino-mediated Camel loss-of-function. The Camel mRNA-mediated gain-of-function causes the Reissner fiber misdirection. This study revealed a link between Chl1a/Camel and Reissner fiber formation, and this supports the idea that CHL1 is one of the scoliosis factors.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Wnt3 distribution in the zebrafish brain is determined by expression, diffusion and multiple molecular interactions.

Wnt3 proteins are lipidated and glycosylated signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG, and Lrp5-mediated receptor binding, to regulate zebrafish brain development.

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/ß-catenin Signaling Regulation.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/ß-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease.

Origin and development of circumventricular organs in living vertebrate.

The circumventricular organs (CVOs) function by mediating chemical communication between blood and brain across the blood-brain barrier. Their origin and developmental mechanisms involved are not understood in enough detail due to a lack of molecular markers common for CVOs. These rather small and inconspicuous organs are found in close vicinity to the third and fourth brain ventricles suggestive of ancient evolutionary origin. Recently, an integrated approach based on analysis of CVOs development in the enhancer-trap transgenic zebrafish led to an idea that almost all of CVOs could be highlighted by GFP expression in this transgenic line. This in turn suggested that an enhancer along with a set of genes it regulates may illustrate the first common element of developmental regulation of CVOs. It seems to be related to a mechanism of suppression of the canonical Wnt/ ß-catenin signaling that functions in development of fenestrated capillaries typical for CVOs. Based on that observation the common molecular elements of the putative developmental mechanism of CVOs will be discussed in this review.

Kv2.1 voltage-gated potassium channels in developmental perspective.

Kv2.1 voltage-gated potassium channels consist of two types of α-subunits: (a) electrically-active Kcnb1 α-subunits and (b) silent or modulatory α-subunits plus ß-subunits that, similar to silent α-subunits, also regulate electrically-active subunits. Voltage-gated potassium channels were traditionally viewed, mainly by electrophysiologists, as regulators of the electrical activity of the plasma membrane in excitable cells, a role that is performed by transmembrane protein domains of α-subunits that form the electric pore. Genetic studies revealed a role for this region of α-subunits of voltage-gated potassium channels in human neurodevelopmental disorders, such as epileptic encephalopathy. The N- and C-terminal domains of α-subunits interact to form the cytoplasmic subunit of heterotetrameric potassium channels that regulate electric pores. Subsequent animal studies revealed the developmental functions of Kcnb1-containing voltage-gated potassium channels and illustrated their role during brain development and reproduction. These functions of potassium channels are discussed in this review in the context of regulatory interactions between electrically-active and regulatory subunits.

A novel conserved enhancer at zebrafish zic3 and zic6 loci drives neural expression.

BACKGROUND: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. RESULTS: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. CONCLUSION: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression.

The translational regulation of maternal mRNAs in time and space.

Since their discovery, the study of maternal mRNAs has led to the identification of mechanisms underlying their spatiotemporal regulation within the context of oogenesis and early embryogenesis. Following synthesis in the oocyte, maternal mRNAs are translationally silenced and sequestered into storage in cytoplasmic granules. At the same time, their unique distribution patterns throughout the oocyte and embryo are tightly controlled and connected to their functions in downstream embryonic processes. At certain points in oogenesis and early embryogenesis, maternal mRNAs are translationally activated to perform their functions in a timely manner. The cytoplasmic polyadenylation machinery is responsible for the translational activation of maternal mRNAs, and its role in initiating the maternal to zygotic transition events has recently come to light. Here, we summarize the current knowledge on maternal mRNA regulation, with particular focus on cytoplasmic polyadenylation as a mechanism for translational regulation.