Biology subjects

Krstevski, A.

Publications and source records attributed to Krstevski, A..

3 recordsLinked to original sources

bioRxiv2025

Patient-specific iPSC models of neural tube defects identify underlying deficiencies in neuroepithelial cell shape regulation and differentiation

Spina bifida and anencephaly are neural tube defects caused by failure of embryonic neural tube closure. Successful closure requires apicobasal elongation and apical constriction of neuroepithelial cells. These critical behaviours have not been studied in human, patient-specific models. We characterise a human iPSC-derived neuroepithelial morphogenesis model that is highly reproducible across three parental iPSC lines of diverse origin and reprogramming technologies. Differentiated neuroepithelial cells actively undergo ROCK-dependent apical constriction. ROCK acts downstream of planar cell polarity/VANGL2 in other species. We find that VANGL2 is up-regulated and phosphorylated during iPSCs-to-neuroepithelial differentiation. The patient-identified VANGL2-R353C mutation does not alter VANGL2 expression, localisation or phosphorylation, but reduces myosin-II phosphorylation and apical constriction relative to congenic control iPSCs. Non-congenic comparisons and forward genetic testing are also informative in this reproducible model. We compare lines reprogrammed from amniocytes of two patients with spina bifida, versus two controls. One patient-derived line forms a morphologically normal neuroepithelium, but fails to differentiate into neurons. The second fails to undergo apicobasal elongation, and harbours compound heterozygous mutations in the MED24 gene previously implicated in neuroepithelial elongation in mice. Thus, iPSC-derived neuroepithelial modelling reveals mechanistic insights into conserved cell behaviours, including apical constriction, apicobasal elongation and neural differentiation, links genetically-impaired apical constriction to human disease, and establishes patient-specific models which recapitulate failures across these heterogenous neuroepithelial functions.

developmental biology↗

bioRxiv2023

CFL1-dependent dynamicity of surface ectoderm filopodia-like protrusions increases neurulation zippering speed in mice

Epithelial fusion is critical for formation of many embryonic tissues. The developing mammalian spinal cord fuses by zippering, through cell behaviours dependent on F-actin turnover. We propose a caudal-to-rostral sequence of surface ectoderm cell behaviours which drive zippering progression in mice, and test the requirement for the F-actin severing protein CFL1 in this process. Key zipper-advancing behaviours are i. constriction of supracellular actomyosin cables; ii. extension of short-lived (<2 min) filopodial or more persistent lamellipodial protrusions, which we live-image establishing pioneering contacts across the midline; remodelling of iii. cell-cell and iv. cell-ECM adhesions; v. zipper advancement through constriction of leading-edge cell borders. In wildtype embryos CFL1 is enriched at the leading edge of surface ectoderm cells adjacent to the zippering point and localised within some protrusions. Conditional Cfl1 deletion in the surface ectoderm produces more prominent F-actin stress fibres, makes filopodial protrusions excessively stable, diminishes their exploratory movements which may establish nascent contacts, and impairs junctional constriction of cells at the point of fusion. Zippering speed is reduced [~]30% in conditional Cfl1-deleted embryos and [~]30% develop spina bifida. We therefore propose that impaired filopodial dynamicity limits the sequence of cell behaviours driving spinal zippering in mice, predisposing to spina bifida.

developmental biology↗

bioRxiv2023

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice

Closed spinal dysraphisms are poorly understood neurodevelopmental malformations commonly classed as neural tube defects. Several, including terminal myelocystocele, selectively affect the distal lumbosacral spine. We previously identified a neural tube closure-initiating point, Closure 5, involved in forming the distal spine of mice. Here we document equivalent morphology of the caudal-most end of the closing posterior neuropore (PNP) in mice and humans, suggesting Closure 5 is conserved in humans. It forms in a region of active fibroblast growth factor (FGF) signalling and pharmacological blockade of FGF receptors (Fgfr) impairs Closure 5 formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre similarly impairs Closure 5 formation and leads to morphologically abnormal PNPs, which nonetheless achieve delayed closure although delayed. After PNP closure, a localised region of the distal neural tube of Fgfr1-disrupted embryos re-opens into a trumpet-like flared central canal between the presumptive hindlimbs, progressing to form a distal fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis of spinal progenitor domains reveals regional and progressive loss of ventral spinal cord progenitor domains preceding cystic dilation of the central canal. Initially, the Shh and FoxA2-positive ventral domains are lost, resulting in Olig2-labelling of the ventral-most neural tube. The Olig2-domain is also subsequently lost, eventually producing a neural tube entirely positive for the dorsal marker Pax3. Thus, a terminal myelocystocele-like phenotype can arise after completion of neural tube closure due to localised spinal mis-patterning caused by disruption of Fgfr1 signalling.

developmental biology↗