Reemployment of Kupffer's vesicle cells into axial and paraxial mesoderm via transdifferentiation

Kupffer's vesicle (KV) in the teleost embryo is a fluid-filled vesicle surrounded by a layer of epithelial cells with rotating primary cilia. KV transiently acts as the left-right organizer but degenerates after the establishment of left-right asymmetric gene expression. Previous labelling experiments indicated that descendants of KV-epithelial cells are incorporated into mesodermal tissues after KV collapses (KV-collapse) in zebrafish embryos. However, the overall picture of their differentiation potency had been unclear due to the lack of suitable genetic tools and molecular analyses. In the present study, we established a novel zebrafish transgenic line with a promoter of charon, in which all KV-epithelial cells and their descendants are specifically labelled until the larval stage. We found that KV-epithelial cells underwent epithelial-mesenchymal transition upon KV-collapse and infiltrate into adjacent mesodermal progenitors, the presomitic mesoderm and chordoneural hinge. Once incorporated, the descendants of KV-epithelial cells expressed distinct mesodermal differentiation markers and contributed to the mature populations such as the axial muscles and notochordal sheath through normal developmental process. These results indicate that fully differentiated KV-epithelial cells possess unique plasticity in that they are reemployed into mesodermal lineages through transdifferentiation after they complete their initial role in KV.

Morphological features of the Black Sea turbot (Scophthalmus maeoticus) during the period of embryonic development

Black Sea turbot (hereinafter BST), Scophthalmus maeoticus (Pallas, 1814), is a valuable fish for commercial fishery and promising object of industrial mariculture. Potential fecundity of BST is very high, 3–13 million eggs; however, survival of its progenies during early development in the sea is unpredictable and low (mortality is up to 90 %). In nature fertilized pelagic BST eggs rise to the sea surface in 2–3 hours; BST develop in upper waters being part of neuston till hatching. BST on its early stages of development could be considered the most vulnerable as the embryo is exposed to diverse adverse effects. The survival and physiological state of the larvae at hatching till exogenous feeding depend on the norm of morphological characteristics of the embryos during their development. Our aim was to study the norm of the changes in BST morphological characteristics during embryogenesis. Morphological analysis of the BST embryogenesis stages from fertilization till hatching on the basis of detailed study of intact embryos (> 2000 eggs) sampled from different experimental batches incubated under experimental conditions is presented. Digital photos and videos of alive eggs were taken with Canon PowerShot A720 using binocular microscope MBS-10 at magnification 8×4 and under light inverted microscope Nikon Eclipse TS100, equipped with analog camera, at magnification ×4, ×10, and ×40. The morphological features of embryogenesis in BST before and after fertilization, cleavage, blastulation, gastrulation, epiboly, and neurulation and until hatching are presented by photos with detailed description of transforming embryological structures. Fertilized pelagic BST eggs covered by transparent shell vary from (1.26 ± 0.14) to (1.31 ± 0.15) mm in diameter, have homogenously distributed yolk and a single round transparent oil drop of 0.20–0.21 mm, positioned at the top of the yolk. Scale of timing of morphological changes is presented in relative time units (as a time interval from fertilization until the emergence of morphological structure in percentage of the total duration of embryogenesis, % RT). Cleavage starts at 2.5 % RT. Cell division desynchronizes between the 6th and 7th cleavage, at 128 blastomeres. Yolk syncytial layer controlling processes of epiboly, cells differentiation, and morphogenesis is formed during the 10th–11th mitotic cycle (12 % RT, about 512–1024 cells). From the germ ring registered at 21 % RT, the embryonic shield develops (at 25 % RT), and organize formation of embryonic axis from 20 to 50 % epiboly (31 % RT). During 70–75 % epiboly (40–45 % RT), the neural keel is formed; notochord and optical primordia become visible; Kupffer’s vesicle emerges at the start of segmentation. Optic cups develop, and more than 20 somites are observed at the end of epiboly (49 % RT). By 60 % RT the Kupffer’s vesicle disappears in tail bud formed; lens placodes are formed in optic cups. Notochord vacuolization, myotomes formation, and tail growth are observed by 65 % RT. The caudal part of the body separates from the yolk by 70–75 % RT. About 80 % RT neuromuscular activity starts; heart beating initiates; free tail covers more than 60 % of the yolk; differentiating xantophores give a pinkish hue to the embryo. By 90–95 % RT eye cups with lenses; three symmetric otic capsules with otoliths, melanophores, and xantophores present in the embryo with 33–38 body somites; it performs jerky movements. Prior hatching, the egg shell becomes elastic, stretches, and breaks in the head area. Hatching occurs 114–94 hours after fertilization at +14…+16 °С. By hatching, all organs are formed in bilateral symmetrical BST larva (standard length is (2.53 ± 0.13) to (2.91 ± 0.10) mm), three auditory chambers with otoliths exist, eyes are non-pigmented, intestinal tract is closed; within 3–5 days it develops at the expense of yolk. Description of morphological changes in the BST embryo at norm of development could be used for elaboration of criteria of developing BST eggs both in natural environment and under cultivation conditions.

Chemosensing versus mechanosensing in nodal and Kupffer's vesicle cilia and in other left–right organizer organs

How is sensing carried out by cilia in the mouse node, zebrafish Kupffer's vesicle and similar left–right (LR) organizer organs in other species? Two possibilities have been put forward. In the former, cilia would detect some chemical species in the fluid; in the latter, they would detect fluid flow. In either case, the hypothesis is that an imbalance would be detected between this signalling coming from cilia on the left and right sides of the organizer, which would initiate a cascade of signals leading ultimately to the breaking of LR symmetry in the developing body plan of the organism. We review the evidence for both hypotheses. This article is part of the Theo Murphy meeting issue ‘Unity and diversity of cilia in locomotion and transport’.

On the Necessary Conditions for Non-Equivalent Solutions of the Rotlet-Induced Stokes Flow in a Sphere: Towards a Minimal Model for Fluid Flow in the Kupffer’s Vesicle

The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserved fundamental process in developmental biology. Details of how symmetry breaking is established in different organisms are, however, still not fully understood. In the zebrafish (Danio rerio), it is known that a cilia-mediated vortical flow exists within its LR organizer, the so-called Kupffer’s vesicle (KV), and that it is directly involved in early LR determination. However, the flow exhibits spatio-temporal complexity; moreover, its conversion to asymmetric development has proved difficult to resolve despite a number of recent experimental advances and numerical efforts. In this paper, we provide further theoretical insight into the essence of flow generation by putting together a minimal biophysical model which reduces to a set of singular solutions satisfying the imposed boundary conditions; one that is informed by our current understanding of the fluid flow in the KV, that satisfies the requirements for left–right symmetry breaking, but which is also amenable to extensive parametric analysis. Our work is a step forward in this direction. By finding the general conditions for the solution to the fluid mechanics of a singular rotlet within a rigid sphere, we have enlarged the set of available solutions in a way that can be easily extended to more complex configurations. These general conditions define a suitable set for which to apply the superposition principle to the linear Stokes problem and, hence, by which to construct a continuous set of solutions that correspond to spherically constrained vortical flows generated by arbitrarily displaced infinitesimal rotations around any three-dimensional axis.

Using zebrafish to study the function of nephronophthisis and related ciliopathy genes

Zebrafish are a valuable vertebrate model in which to study development and characterize genes involved in cystic kidney disease. Zebrafish embryos and larvae are transparent, allowing non-invasive imaging during their rapid development, which takes place over the first 72 hours post fertilisation. Gene-specific knockdown of nephronophthisis-associated genes leads to ciliary phenotypes which can be assessed in various developmental structures. Here we describe in detail the methods used for imaging cilia within Kupffer’s vesicle to assess nephronophthisis and related ciliopathy phenotypes.

Using zebrafish to study the function of nephronophthisis and related ciliopathy genes

Zebrafish are a valuable vertebrate model in which to study development and characterize genes involved in cystic kidney disease. Zebrafish embryos and larvae are transparent, allowing non-invasive imaging during their rapid development, which takes place over the first 72 hours post fertilisation. Gene-specific knockdown of nephronophthisis-associated genes leads to ciliary phenotypes which can be assessed in various developmental structures. Here we describe in detail the methods used for imaging cilia within Kupffer’s vesicle to assess nephronophthisis and related ciliopathy phenotypes.