Biology subjects

Korzh, V.

Publications and source records attributed to Korzh, V..

8 recordsLinked to original sources

bioRxiv2025

From patterning to secretion: Kv2.1 subunits as regulators of zebrafish hatching gland morphogenesis and function

Zebrafish hatching, a critical developmental milestone, occurs around 48-72 hours post-fertilization (hpf). It is regulated by the specialized secretory organ called the hatching gland. Voltage-gated potassium channels (Kv) are known for their roles in maintaining plasma membrane potential and regulating intracellular protein traffic and secretion. Previous studies on zebrafish mutants of Kv2.1 channel subunits - the electrically active subunit Kcnb1 and the modulatory subunit Kcng4b - revealed antagonistic functions in the development of brain ventricles, ear, and Reissner fiber. In this study, we investigated their functional role in the hatching gland. The loss of either subunit resulted in a significant delay in normal hatching. Using in situ hybridization and immunohistochemistry, we show that both mutants exhibited severe defects in the hatching gland patterning, including a reduced number of hatching gland cells. The mutants displayed changes in the transcript levels of several hatching gland markers and reduced cell proliferation in this organ. These developmental defects were intensified by a late-stage functional failure characterized by decreased cathepsin synthesis, reduced proteolytic activity, and delay in the period of secretion in both mutants. Together, our findings establish that Kv2.1 subunits, Kcnb1, and Kcng4b are essential during the development of the zebrafish hatching gland and its secretion.

developmental biology↗

bioRxiv2025

Kv2.1-Kv6.4 subunits deficiency impairs inhibitory signaling and visual circuit dynamics in zebrafish

Voltage-gated potassium channels (Kv) play a crucial role in maintaining the cells resting potential. Mutations in the Kv2.1 voltage-gated potassium channel are associated with developmental epileptic encephalopathy. Previous analysis of the loss-of-function zebrafish mutants kcnb1sq301 and kcng4bwaw304, which affect genes that encode Kv2.1 subunits (the subunit Kcnb1 and the modulatory subunit Kcng4b), has shown that they play an antagonistic role in the development of hollow organs, such as the brain and ear. In this study, we investigated the behavioral effects of these mutations. Under normal light conditions, both mutants exhibited reduced activity at 5 days post-fertilization. However, exposure to a low concentration of 5 mM of the chemoconvulsant pentylenetetrazole increased their locomotor activity and induced seizures. Quantitative RT-PCR (qRT-PCR) analysis of the mutants revealed an increase in the transcript levels of c-fos and gad2, and a decrease in a transcript level of gabra1. This suggests that mutations cause a disruption to inhibitory neurotransmission. Local field potential recordings from the optic tectum of the mutants under baseline conditions showed an increase in spontaneous electrical activity. The kcnb1 mutant is photosensitive and experiences freezing episodes under high-intensity light. Together, these findings suggest that defects in Kv2.1 subunits impact both locomotor behavior and light-evoked responses.

developmental biology↗

bioRxiv2024

Kcnb1-Kcng4 axis regulates Scospondin secretion and Reissner fiber development

The voltage-gated potassium channel Kv2.1 plays a role in the development of the ventricular system and the subcommissural organ in zebrafish. Here, a role for Kv2.1 in the secretion of the major component of Reissners fiber, Scospondin, was demonstrated. The results showed that Kv2.1 acts as a negative regulator of Scospondin secretion and Reissner fiber assembly. Kv2.1 regulates formation of Scospondin microfilaments and their assembly in Reissner fiber. Cholesterol playing a key role in Scospondin secretion. After the Reissner fiber is formed, it is detached from the hindbrain floor plate, where Scospondin produced initially. The tension of the fiber depends on its attachment to the subcommissural and flexural organs. In turn fiber tension affects the morphogenesis of these organs. This process of Reissner fiber formation depends on the input provided by the Hedgehog and Wnt/{beta}-catenin signaling pathways on the anterior roof and floor plates.

developmental biology↗

bioRxiv2023

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 tetramer consists of an electrically active subunit (Kcnb1, Kv2.1) and a modulatory silent subunit (Kcng4b, Kv6.4). The two mutations in zebrafish kcng4b - 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.

developmental biology↗

bioRxiv2021

Transcriptome profile of the zebrafish atrioventricular canal reveals molecular signatures of pacemaker and valve mesenchyme

The atrioventricular canal (AVC) is an essential feature of the heart, which separates the atrium from the ventricle. During heart morphogenesis, it is a hub of molecular processes necessary for distinguishing heart regions; most importantly, for the formation of the AV conduction system and cardiac valves. To better understand the molecular processes underlying AVC development and function, we utilized the transgenic zebrafish line sqet31Et with EGFP expression in the AVC region to isolate this cell population by FACS and profiled its transcriptome by RNA-seq at 48 and 72 hours post fertilization (hpf). Compared to the rest of the heart, the AVC is enriched for the expression of molecular markers associated with mammalian AVC and AV node, including cx36.7 and cx45 which encode connexins forming low conductance gap junctions. Using the transgenic line Tg(myl7:mermaid) encoding the voltage-sensitive fluorescent protein, we showed that loss of function of Isl1 abolished the pacemaker-containing sinoatrial ring (SAR) and resulted in an erratic spread of excitation pattern from the SAR to AVC, indicating the dysfunction of the primary pacemaker. Concurrently, ectopic excitation in the AVC region was observed, suggesting that the zebrafish AVC possesses inherent automaticity although insufficient to replace the primary pacemaking activity of the SAR. Comparisons between the SAR and AVC transcriptomes revealed partially overlapping expression profiles of various ion channels and gap junction proteins which reflects their diversified functions. Lastly, we observed dynamic expression of epithelial-to-mesenchymal transition markers, as well as components of TGF-{beta}, Notch, and Wnt signaling pathways, which have been implicated in the formation of AVC conduction and cardiac valves. Our results uncovered the molecular hallmarks of the developing AVC region and demonstrated its role in the structural and electrophysiological separation between the atrium and ventricle. Author summaryThe atrioventricular canal is a structure in the embryonic heart which separates the atrium from the ventricle. It gives rise to the AV node and cardiac valves - two important structures which ensure unidirectional blood flow between heart chambers. The AV node serves to regulate the propagation of electrical impulses between the two chambers, such that they contract consecutively. Using the zebrafish as model organism, we performed gene expression profiling and characterized electrical conduction patterns between the sinoatrial primary pacemaker and AVC. We discovered that the zebrafish AVC possesses similar features to the mammalian AV node, including slow conduction, inherent pacemaking activity, and the expression of conserved developmental genes. The molecular profile of the AVC is distinct from that of the sinoatrial pacemaker, which reflects their distinct roles. In addition, we found that genes regulating cardiac valve development were also expressed in the AVC, illustrating the importance of this region for establishing both electrophysiological and structural separation between the heart chambers. Besides establishing conserved aspects between zebrafish and mammalian conduction system, the data generated in this study constitutes a valuable resource for studying AVC development and discovery of novel candidate genes implicated in regulating cardiac rhythm and cardiac valve formation.

developmental biology↗

bioRxiv2020

High behavioural 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. Yet, the function 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-/- 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. We demonstrate that this is due to the reduced excitability of Mauthner neurons resulting in unreliable firing with stimuli that normally elicit the escape response. Combined with previous studies that show Auts2-regulation of the transcription of ion channel proteins, our results suggest that Auts2 sets the excitability of neurons by activating a set transcriptional program. Significance statementAUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to the large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

neuroscience↗

bioRxiv2020

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

Wnt3 proteins are lipidated and glycosylated, secreted 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 photo-bleaching. 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.

developmental biology↗

bioRxiv2020

Evolutionary context can clarify teleosts gene names

The initial convention to name genes relied on historical precedent, order in the human genome or mutants in model systems. However, partial duplication of genes in teleosts required naming the duplicated genes, so ohnologs adopted the a or b extension. Rapid advances in deciphering the zebrafish genome in relation to the human genome instituted naming genes in all other fish genomes in the convention of zebrafish. Unfortunately, some ohnologs and their resembling orthologs suffered from incorrect nomenclature, which created confusion in particular instances like establishing disease models. We sought to overcome this barrier by establishing the ex silico evolutionary-based systematic approach to naming the ohnologs in teleosts and other fish. We compared gene synteny using the spotted gar genome as the reference, which represents the unduplicated ancestral state. Using new criteria, we identified several hundreds of potentially misnamed ohnologs and validated manually several ohnologs as a proof pf principle. This may help to establish a standard naming practice resulting in the improved evolutionary-based gene nomenclature. This approach may help to identify and rename ohnologs in all relevant EMBL-EBI and NCBI databases starting from the zebrafish genome to avoid further proliferation of misleading information.

genomics↗