Defects In Morphogenesis Research Articles

Planar cell polarity zebrafish models of congenital scoliosis reveal novel underlying defects in notochord morphogenesis.

Congenital scoliosis (CS) is a type of vertebral malformation whose etiology remains elusive. The notochord is pivotal for vertebrae development but its role in CS is still understudied. Zebrafish knockout of ptk7a, a planar cell polarity (PCP) gene that is essential for convergence and extension (C&E) of the notochord, developed congenital scoliosis-like vertebral malformations (CVM). Maternal zygotic ptk7a mutants displayed severe C&E defects of the notochord. Excessive apoptosis occurred in the malformed notochord, causing a significantly reduced number of vacuolated cells, and compromising the mechanical properties of the notochord. The latter manifested as a less stiff extracellular matrix along with a significant reduction in the number of the caveolae and severely loosened intercellular junctions in the vacuolated region. These defects led to focal kinks, abnormal mineralization, and CVM exclusively at the anterior spine. Loss of function of another PCP gene, vangl2, also revealed excessive apoptosis in the notochord associated with CVM. This study suggests a new model for CS pathogenesis that is associated with defects in notochord C&E and highlights an essential role of PCP signaling in vertebrae development.

Polymelia and concurrent intestinal duplication and cloacal atresia in a broiler chick: case report and literature review.

Congenital malformations are a highly diverse group of conditions reported in both humans and animals, characterized by defects in morphogenesis observed at birth. Although most cases are idiopathic, genetic and environmental factors may be involved. The frequency of such conditions varies with species, geographic regions, and the specific malformation involved. In polymelia, supernumerary limbs are attached to different parts of the body. Gastrointestinal duplications are described less frequently and can be associated with polymelia. Cloacal atresia is among the least-reported malformations in avian species, described only once in a kiwi. Here we describe a case with these 3 malformations in a single broiler chick (Gallus gallus domesticus) and provide a literature review about the occurrence of these malformations in birds. The 3-d-old chick also had an unidentified structure projecting from the pygostyle region. We performed clinical, radiographic, and postmortem examinations. The intestinal duplication was identified only during the postmortem evaluation. Detailed descriptions of avian congenital malformations are scarce. Although similar cases have been reported, we retrieved no cases of concurrent polymelia, intestinal duplication, and cloacal atresia in broiler chickens in our literature search, suggesting that the simultaneous occurrence of these conditions has not been reported previously in this species.

Apoptosis-dependent head development during metamorphosis of the cnidarian Hydractinia symbiolongicarpus

Apoptosis is a regulated cell death that depends on caspases. It has mainly been studied as a mechanism for the removal of unwanted cells. However, apoptotic cells can induce fate or behavior changes of their neighbors and thereby participate in development. Here, we address the functions of apoptosis during metamorphosis of the cnidarian Hydractinia symbiolongicarpus. We describe the apoptotic profile during metamorphosis of the larva and identify Caspase3/7a, but no other executioner caspases, as essential for apoptosis in this context. Using pharmacological and genetic approaches, we find that apoptosis is required for normal head development. Inhibition of apoptosis resulted in defects in head morphogenesis. Neurogenesis was compromised in the body column of apoptosis-inhibited animals but there was no effect on the survival or proliferation of stem cells, suggesting that apoptosis is required for cellular commitment rather than for the maintenance of their progenitors. Differential transcriptomic analysis identifies TRAF genes as downregulated in apoptosis-inhibited larvae and functional experiments provide evidence that they are essential for head development. Finally, we find no major role for apoptosis in head regeneration in this animal, in contrast to the significance of apoptosis in Hydra head regeneration.

Inherited mitochondrial dysfunction triggered by OPA1 mutation impacts the sensory innervation fibre identity, functionality and regenerative potential in the cornea

Mitochondrial dysfunctions are detrimental to organ metabolism. The cornea, transparent outmost layer of the eye, is prone to environmental aggressions, such as UV light, and therefore dependent on adequate mitochondrial function. While several reports have linked corneal defects to mitochondrial dysfunction, the impact of OPA1 mutation, known to induce such dysfunction, has never been studied in this context. We used the mouse line carrying OPA1delTTAG mutation to investigate its impact on corneal biology. To our surprise, neither the tear film composition nor the corneal epithelial transcriptomic signature were altered upon OPA1 mutation. However, when analyzing the corneal innervation, we discovered an undersensitivity of the cornea upon the mutation, but an increased innervation volume at 3 months. Furthermore, the fibre identity changed with a decrease of the SP + axons. Finally, we demonstrated that the innervation regeneration was less efficient and less functional in OPA1+/- corneas. Altogether, our study describes the resilience of the corneal epithelial biology, reflecting the mitohormesis induced by the OPA1 mutation, and the adaptation of the corneal innervation to maintain its functionality despite its morphogenesis defects. These findings will participate to a better understanding of the mitochondrial dysfunction on peripheral innervation.

An incoherent feed-forward loop involving bHLH transcription factors, Auxin and CYCLIN-Ds regulates style radial symmetry establishment in Arabidopsis.

The bilateral-to-radial symmetry transition occurring during the development of the Arabidopsis thaliana female reproductive organ (gynoecium) is a crucial biological process linked to plant fertilization and seed production. Despite its significance, the cellular mechanisms governing the establishment and breaking of radial symmetry at the gynoecium apex (style) remain unknown. To fill this gap, we employed quantitative confocal imaging coupled with MorphoGraphX analysis, in vivo and in vitro transcriptional experiments, and genetic analysis encompassing mutants in two bHLH transcription factors necessary and sufficient to promote transition to radial symmetry, SPATULA (SPT) and INDEHISCENT (IND). Here, we show that defects in style morphogenesis correlate with defects in cell-division orientation and rate. We showed that the SPT-mediated accumulation of auxin in the medial-apical cells undergoing symmetry transition is required to maintain cell-division-oriented perpendicular to the direction of organ growth (anticlinal, transversal cell division). In addition, SPT and IND promote the expression of specific core cell-cycle regulators, CYCLIN-D1;1 (CYC-D1;1) and CYC-D3;3, to support progression through the G1 phase of the cell cycle. This transcriptional regulation is repressed by auxin, thus forming an incoherent feed-forward loop mechanism. We propose that this mechanism fine-tunes cell division rate and orientation with the morphogenic signal provided by auxin, during patterning of radial symmetry at the style.

PDX1+ cell budding morphogenesis in a stem cell-derived islet spheroid system

Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches, we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis, the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture, accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development, we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach, we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases.

Isl Identifies the Extraembryonic Mesodermal/Allantois Progenitors and is Required for Placenta Morphogenesis and Vasculature Formation.

The placenta links feto-maternal circulation for exchanges of nutrients, gases, and metabolic wastes between the fetus and mother, being essential for pregnancy process and maintenance. The allantois and mesodermal components of amnion, chorion, and yolk sac are derived from extraembryonic mesoderm (Ex-Mes), however, the mechanisms contributing to distinct components of the placenta and regulation the interactions between allantois and epithelium during chorioallantoic fusion and labyrinth formation remains unclear. Isl1 is expressed in progenitors of the Ex-Mes and allantois the Isl1 mut mouse line is analyzed to investigate contribution of Isl1+ Ex-Mes / allantoic progenitors to cells of the allantois and placenta. This study shows that Isl1 identifies the Ex-Mes progenitors for endothelial and vascular smooth muscle cells, and most of the mesenchymal cells of the placenta and umbilical cord. Deletion of Isl1 causes defects in allantois growth, chorioallantoic fusion, and placenta vessel morphogenesis. RNA-seq and CUT&Tag analyses revealed that Isl1 promotes allantoic endothelial, inhibits mesenchymal cell differentiation, and allantoic signals regulated by Isl1 mediating the inductive interactions between the allantois and chorion critical for chorionic epithelium differentiation, villous formation, and labyrinth angiogenesis. This study above reveals that Isl1 plays roles in regulating multiple genetic and epigenetic pathways of vascular morphogenesis, provides the insight into the mechanisms for placental formation, highlighting the necessity of Isl1 for placenta formation/pregnant maintenance.

Ankyrin-B is required for the establishment and maintenance of lens cytoarchitecture, mechanics, and clarity.

This study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.

RBX1-CRLs Promote Perinatal Heart Development by Regulating Hippo-YAP Signaling

Background: During development, the heart is the first organ to form, ensuring the pumping of blood through the body. Defects in cardiac morphogenesis can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms underlying this process remain incompletely understood. Cullin-RING E3 ubiquitin ligases (CRLs) are multi-subunit complexes critical to maintaining protein homeostasis by targeting diverse protein substrates for ubiquitination and subsequent degradation. In CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Germline ablation of RBX1 in worms, flies, and mice caused embryonic lethality. Deletion of RBX1 in zebrafish resulted in cardiac malformation and dysfunction, which was rescued by endothelial, but not myocardial, RBX1 overexpression. However, the cardiomyocyte-autonomous role of RBX1 in the heart in vertebrates is unknown. Therefore, we hypothesize that cardiomyocyte RBX1 is indispensable for cardiac development. Methods and Results: We generated a cardiomyocyte-specific RBX1 knockout (RBX1CKO) mouse model via αMHCCre. RBX1-deficient mouse embryos were initially morphologically indistinguishable from the littermate controls at E14.5, but began to show peripheral edema and hemorrhage at E16.5, and eventually died around E18.5. Histological analysis of RBX1CKO hearts revealed severe myocardial hypoplasia and bi-ventricular non-compaction by E14.5. Bulk RNA sequencing and proteomic analyses of RBX1CKO hearts identified perturbations in glycolytic to oxidative transition and dysregulation of sarcomeric proteins. EdU incorporation assay showed a significant reduction in cardiomyocyte proliferation in E14.5 RBX1-deficient hearts, accompanied by the impaired nuclear translocation of YAP, a co-transcriptional factor essential for cardiomyocyte proliferation and maturation. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1, leading to the elevated phosphorylation and inactivation of YAP. We further demonstrated that loss of RBX1 inhibited the degradation of the Hippo adaptor MOB1, likely due to suppressed ubiquitination. Conclusion: Our study identifies RBX1 as an essential regulator of ventricular maturation and found that RBX1-mediated proteolysis of Hippo kinases serves as a critical checkpoint to ensure YAP activation and, hence, cardiomyocyte proliferation and maturation during cardiac development. AHA Predoctoral Fellowship, American Heart Association, 23PRE1012641. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Buffering Mechanism in Aortic Arch Artery Formation and Congenital Heart Disease.

The resiliency of embryonic development to genetic and environmental perturbations has been long appreciated; however, little is known about the mechanisms underlying the robustness of developmental processes. Aberrations resulting in neonatal lethality are exemplified by congenital heart disease arising from defective morphogenesis of pharyngeal arch arteries (PAAs) and their derivatives. Mouse genetics, lineage tracing, confocal microscopy, and quantitative image analyses were used to investigate mechanisms of PAA formation and repair. The second heart field (SHF) gives rise to the PAA endothelium. Here, we show that the number of SHF-derived endothelial cells (ECs) is regulated by VEGFR2 (vascular endothelial growth factor receptor 2) and Tbx1. Remarkably, when the SHF-derived EC number is decreased, PAA development can be rescued by the compensatory endothelium. Blocking such compensatory response leads to embryonic demise. To determine the source of compensating ECs and mechanisms regulating their recruitment, we investigated 3-dimensional EC connectivity, EC fate, and gene expression. Our studies demonstrate that the expression of VEGFR2 by the SHF is required for the differentiation of SHF-derived cells into PAA ECs. The deletion of 1 VEGFR2 allele (VEGFR2SHF-HET) reduces SHF contribution to the PAA endothelium, while the deletion of both alleles (VEGFR2SHF-KO) abolishes it. The decrease in SHF-derived ECs in VEGFR2SHF-HET and VEGFR2SHF-KO embryos is complemented by the recruitment of ECs from the nearby veins. Compensatory ECs contribute to PAA derivatives, giving rise to the endothelium of the aortic arch and the ductus in VEGFR2SHF-KO mutants. Blocking the compensatory response in VEGFR2SHF-KO mutants results in embryonic lethality shortly after mid-gestation. The compensatory ECs are absent in Tbx1+/- embryos, a model for 22q11 deletion syndrome, leading to unpredictable arch artery morphogenesis and congenital heart disease. Tbx1 regulates the recruitment of the compensatory endothelium in an SHF-non-cell-autonomous manner. Our studies uncover a novel buffering mechanism underlying the resiliency of PAA development and remodeling.

Altered neuroepithelial morphogenesis and migration defects in iPSC-derived cerebral organoids and 2D neural stem cells in familial bipolar disorder.

Bipolar disorder (BD) is a severe mental illness that can result from neurodevelopmental aberrations, particularly in familial BD, which may include causative genetic variants. In the present study, we derived cortical organoids from BD patients and healthy (control) individuals from a clinically dense family in the Indian population. Our data reveal that the patient organoids show neurodevelopmental anomalies, including organisational, proliferation and migration defects. The BD organoids show a reduction in both the number of neuroepithelial buds/cortical rosettes and the ventricular zone size. Additionally, patient organoids show a lower number of SOX2-positive and EdU-positive cycling progenitors, suggesting a progenitor proliferation defect. Further, the patient neurons show abnormal positioning in the ventricular/intermediate zone of the neuroepithelial bud. Transcriptomic analysis of control and patient organoids supports our cellular topology data and reveals dysregulation of genes crucial for progenitor proliferation and neuronal migration. Lastly, time-lapse imaging of neural stem cells in 2D in vitro cultures reveals abnormal cellular migration in BD samples. Overall, our study pinpoints a cellular and molecular deficit in BD patient-derived organoids and neural stem cell cultures.

C. elegans Afadin is required for epidermal morphogenesis and functionally interfaces with the cadherin-catenin complex and RhoGAP PAC-1/ARHGAP21

During epithelial morphogenesis, the apical junctions connecting cells must remodel as cells change shape and make new connections with their neighbors. In the C. elegans embryo, new apical junctions form when epidermal cells migrate and seal with one another to encase the embryo in skin (‘ventral enclosure’), and junctions remodel when epidermal cells change shape to squeeze the embryo into a worm shape (‘elongation’). The junctional cadherin-catenin complex (CCC), which links epithelial cells to each other and to cortical actomyosin, is essential for C. elegans epidermal morphogenesis. RNAi genetic enhancement screens have identified several genes encoding proteins that interact with the CCC to promote epidermal morphogenesis, including the scaffolding protein Afadin (AFD-1), whose depletion alone results in only minor morphogenesis defects. Here, by creating a null mutation in afd-1, we show that afd-1 provides a significant contribution to ventral enclosure and elongation on its own. Unexpectedly, we find that afd-1 mutant phenotypes are strongly modified by diet, revealing a previously unappreciated parental nutritional input to morphogenesis. We identify functional interactions between AFD-1 and the CCC by demonstrating that E-cadherin is required for the polarized distribution of AFD-1 to cell contact sites in early embryos. Finally, we show that afd-1 promotes the enrichment of polarity regulator, and CCC-interacting protein, PAC-1/ARHGAP21 to cell contact sites, and we identify genetic interactions suggesting that afd-1 and pac-1 regulate epidermal morphogenesis at least in part through parallel mechanisms. Our findings reveal that C. elegans AFD-1 makes a significant contribution to epidermal morphogenesis and functionally interfaces with core and associated CCC proteins.

MARK1 regulates dendritic spine morphogenesis and cognitive functions in vivo

Dendritic spines play a pivotal role in synaptic communication and are crucial for learning and memory processes. Abnormalities in spine morphology and plasticity are observed in neurodevelopmental and neuropsychiatric disorders, yet the underlying signaling mechanisms remain poorly understood. The microtubule affinity regulating kinase 1 (MARK1) has been implicated in neurodevelopmental disorders, and the MARK1 gene shows accelerated evolution in the human lineage suggesting a role in cognition. However, the in vivo role of MARK1 in synaptogenesis and cognitive functions remains unknown. Here we show that forebrain-specific conditional knockout (cKO) of Mark1 in mice causes defects in dendritic spine morphogenesis in hippocampal CA1 pyramidal neurons with a significant reduction in spine density. In addition, we found loss of MARK1 causes synaptic accumulation of GKAP and GluA2. Furthermore, we found that MARK1 cKO mice show defects in spatial learning in the Morris water maze and reduced anxiety-like behaviors in the elevated plus maze. Taken together, our data show a novel role for MARK1 in regulating dendritic spine morphogenesis and cognitive functions in vivo.

Acclimation of intertidally reproducing sea-snails protects embryos from lethal effects of transient hyperthermia.

Embryos of Ilyanassa obsoleta (from Massachusetts and Florida) and Phrontis vibex (from Florida) were exposed to temperatures from 33to 37°C. In both species, very young embryos are especially sensitive to thermal stress. Brief early heat shock did not disturb spiral cleavage geometry but led to variable, typically severe defects in larval morphogenesis and tissue differentiation. In Ilyanassa but not P. vibex, early heat shock resulted in immediate slowing or arrest of interphase progression during early cleavage. This reversible arrest was correlated with improved prognosis for larval development and (in Massachusetts snails, at least) depended on parental acclimation to warm temperature (~25.5°C). Embryos from Massachusetts snails housed at lower temperature (16°C) exhibited cytokinesis failure when briefly incubated at 33°C during early cleavage, and tissue differentiation failure during incubation at 33°C begun at later stages. This preliminary study reveals a case in which stress-conditioned parents may endow embryos with protection against potentially lethal thermal stress during the most vulnerable stages of life.

A missense SNP in the tumor suppressor SETD2 reduces H3K36me3 and mitotic spindle integrity in Drosophila.

Mutations in SETD2 are among the most prevalent drivers of renal cell carcinoma (RCC). We identified a novel single nucleotide polymorphism (SNP) in SETD2, E902Q, within a subset of RCC patients, which manifests as both an inherited or tumor-associated somatic mutation. To determine if the SNP is biologically functional, we used CRISPR-based genome editing to generate the orthologous mutation within the Drosophila melanogaster Set2 gene. In Drosophila, the homologous amino acid substitution, E741Q, reduces H3K36me3 levels comparable to Set2 knockdown, and this loss is rescued by reintroduction of a wild-type Set2 transgene. We similarly uncovered significant defects in spindle morphogenesis, consistent with the established role of SETD2 in methylating α-Tubulin during mitosis to regulate microtubule dynamics and maintain genome stability. These data indicate the Set2 E741Q SNP affects both histone methylation and spindle integrity. Moreover, this work further suggests the SETD2 E902Q SNP may hold clinical relevance.

Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis

BackgroundDysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14–3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function.ResultsHere we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner.ConclusionsWe found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.

A New Derivative of Retro-2 Displays Antiviral Activity against Respiratory Syncytial Virus.

Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in newborns, with all children being infected before the age of two. Reinfections are very common throughout life and can cause severe respiratory infections in the elderly and immunocompromised adults. Although vaccines and preventive antibodies have recently been licensed for use in specific subpopulations of patients, there is still no therapeutic treatment commonly available for these infections. Here, we investigated the potential antiviral activity of Retro-2.2, a derivative of the cellular retrograde transport inhibitor Retro-2, against hRSV. We show that Retro-2.2 inhibits hRSV replication in cell culture and impairs the ability of hRSV to form syncytia. Our results suggest that Retro-2.2 treatment affects virus spread by disrupting the trafficking of the viral de novo synthetized F and G glycoproteins to the plasma membrane, leading to a defect in virion morphogenesis. Taken together, our data show that targeting intracellular transport may be an effective strategy against hRSV infection.

Prenatal developmental sequences of the esophageal epithelium in the New Zealand white rabbits: Light and electron microscopic analysis.

Several morphogenetic sequences occur during esophageal development and birth defects occur due to defects in foregut morphogenesis. This work aimed to record the cellular events in the morphogenesis of rabbits' esophageal epithelium. On the 16th day of gestation, the esophageal epithelium varied from stratified ciliated columnar to stratified squamous type. The surface epithelium presented mucous cells with mucigen granules of various sizes occupying their supranuclear cytoplasm. Cytoplasmic vacuolation was evident in all layers of the esophageal epithelium at this age. On the 18th gestational day, some light cells could be detected in the middle portion of the epithelium, while others occupied the whole epithelial length. On the 21st day, mucous cells are more frequently observed at the apical esophageal part as well as at the surface epithelium. Numerous elongated dark cells could be distinguished embedded between the basal cells. On the 24th gestational day the number of the mucous cells reached its peak. Reaching the 30th gestational day, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased. Collectively, detection of surface mucous and dark cells together with the non-cornified surface in some regions of the rabbit esophageal epithelium at the end of gestation ensure a postnatal development to reach the adult epithelium essential to sustain the passage of the harsh raw food. Future immunohistochemical studies are recommended to investigate the components of secretions in mucous cells and functional studies to highlight the dark cells significance. RESEARCH HIGHLIGHTS: Esophageal epithelium of fetal rabbit was analyzed by light and transmission microscopy. Surface epithelium presented mucous cells with mucigen granules of various sizes. They reached their maximum number on 24th day then decreased. On the 16th day, cytoplasmic vacuolation was evident in all epithelial layers. On the 21st day, numerous elongated dark cells could be distinguished embedded between the basal cells. Before birth, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased.

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

Knockout of eight hydroxyproline-O-galactosyltransferases cause multiple vegetative and reproductive growth defects

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of β-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation.