Head Of Embryos Research Articles

Characterization and Spatiotemporal Expression of Klf4 in Large Yellow Croaker Larimichthys crocea.

As one of transcription factors and pluripotency factors, Klf4 plays a crucial role in regulation of cellular processes. In this article, we characterized Klf4 of large yellow croaker (Lc-Klf4), which encodes a 452-amino acid protein (Lc-Klf4) with three highly conserved C2H2 zinc fingers. Lc-Klf4 shares high conservative functions in teleosts with the closest relationship with Stegastes partitus. The spatiotemporal expression showed that Lc-Klf4 was expressed widely in adult tissues with gender difference as follows: brain>gill>eye>heart in female; heart>testis>gill>brain in male; male>female in heart, gill, and testis; and female>male in eye. During growth, the highest expression level of Lc-Klf4 was at 635 dph (days posthatching) in testis and at 270 dph and 635 dph in brain. Besides, Lc-Klf4 was widely and highly distributed in different developmental spermatids especially in spermatocytes. The expression of Lc-Klf4 in embryos was exhibited zygotically beginning from late gastrula stage with high level in closure of blastopore stage and appearance of optic vesicle stage. This expression pattern was supported by whole-mount in situ hybridization with high expression in back and head of late embryos. In conclusion, the spatiotemporal expression patterns of Lc-Klf4 illustrated that Klf4 involves in spermatogenesis, embryogenesis, and adult physiological processes.

Ein In-vitro-Mausmodell für die Palatogenese und die Entstehung von Gaumenspalten

AbstractThe mouse is an excellent model to study palatogenesis since, like humans, it is a mammal, it can easily be bred and a wide array of molecular tools are available. Moreover, the isolated embryonic mouse palate can be cultured in vitro, which allows us to study the actual fusion process in detail.Just prior to the actual fusion at E13, the maxilla with the 2 palatal shelves can be dissected from the embryo head. The maxilla is cultured on a filter paper on top of a stainless-steel grid at the interface of the culture medium and air. After 24 h in culture, the palatal shelves are in contact and the MES is clearly visible. At 48 h, the MES has nearly completely disintegrated with small remnants at the nasal and oral side of the palate. Finally, at 72 h, the palatal shelves are completely fused and osteoid tissue is beginning to form in the lateral areas.The exact downstream molecular mechanisms that lead to clefting are far from being unraveled. The model for palate fusion presented here offers the possibility to analyze the molecular and cellular mechanisms that lead to clefting as a result of specific genetic and environmental factors. This will pave the way to pharmacological intervention for the prevention of cleft lip and/or palate in susceptible individuals.

MORN5 Expression during Craniofacial Development and Its Interaction with the BMP and TGFβ Pathways.

MORN5 (MORN repeat containing 5) is encoded by a locus positioned on chromosome 17 in the chicken genome. The MORN motif is found in multiple copies in several proteins including junctophilins or phosphatidylinositol phosphate kinase family and the MORN proteins themselves are found across the animal and plant kingdoms. MORN5 protein has a characteristic punctate pattern in the cytoplasm in immunofluorescence imaging. Previously, MORN5 was found among differentially expressed genes in a microarray profiling experiment of the chicken embryo head. Here, we provided in situ hybridization to analyse, in detail, the MORN5 expression in chick craniofacial structures. The expression of MORN5 was first observed at stage HH17-18 (E2.5). MORN5 expression gradually appeared on either side of the primitive oral cavity, within the maxillary region. At stage HH20 (E3), prominent expression was localized in the mandibular prominences lateral to the midline. From stage HH20 up to HH29 (E6), there was strong expression in restricted regions of the maxillary and mandibular prominences. The frontonasal mass (in the midline of the face) expressed MORN5, starting at HH27 (E5). The expression was concentrated in the corners or globular processes, which will ultimately fuse with the cranial edges of the maxillary prominences. MORN5 expression was maintained in the fusion zone up to stage HH29. In sections MORN5 expression was localized preferentially in the mesenchyme. Previously, we examined signals that regulate MORN5 expression in the face based on a previous microarray study. Here, we validated the array results with in situ hybridization and QPCR. MORN5 was downregulated 24 h after Noggin and/or RA treatment. We also determined that BMP pathway genes are downstream of MORN5 following siRNA knockdown. Based on these results, we conclude that MORN5 is both regulated by and required for BMP signaling. The restricted expression of MORN5 in the lip fusion zone shown here supports the human genetic data in which MORN5 variants were associated with increased risk of non-syndromic cleft lip with or without cleft palate.

Reiterative expression of pax1 directs pharyngeal pouch segmentation in medaka.

A striking characteristic of vertebrate development is the pharyngeal arches, which are a series of bulges on the lateral surface of the head of vertebrate embryos. Although each pharyngeal arch is segmented by the reiterative formation of endodermal outpocketings called pharyngeal pouches, the molecular network underlying the reiterative pattern remains unclear. Here, we show that pax1 plays crucial roles in pouch segmentation in medaka (Oryzias latipes) embryos. Importantly, pax1 expression in the endoderm prefigures the location of the next pouch before the cells bud from the epithelium. TALEN-generated pax1 mutants did not form pharyngeal pouches posterior to the second arch. Segmental expression of tbx1 and fgf3, which play essential roles in pouch development, was almost non-existent in the pharyngeal endoderm of pax1 mutants, with disturbance of the reiterative pattern of pax1 expression. These results suggest that pax1 plays a key role in generating the primary pattern for segmentation in the pharyngeal endoderm by regulating tbx1 and fgf3 expression. Our findings illustrate the crucial roles of pax1 in vertebrate pharyngeal segmentation and provide insights into the evolutionary origin of the deuterostome gill slit.

Radiation dose to the fetus during CyberKnife radiosurgery for a brain tumor in pregnancy

PurposePregnancy during radiosurgery is extremely rare in clinical practice. We report fetal dose results during CyberKnife radiosurgery for a brain tumor in pregnancy. Methods and materialsA 26 year old pregnant woman with a rapidly growing deep-seated grade-III glioma was treated during the third trimester of gestation using CyberKnife. Ultrasound imaging was used to determine the position of the embryo prior to treatment. A dose of 1400 cGy was prescribed aiming to control tumor growth until delivery of the child. Prior to radiosurgery, the treatment was simulated on an anthropomorphic phantom. Radiation dose to the embryo was measured using a Farmer chamber and EBT3 films. ResultsFetal doses of 4.4 cGy and 4.1 cGy were measured for the embryo's head and legs, lying at 56 cm and 72 cm from the isocenter, respectively, using the Farmer chamber situated at 8.5 cm depth beneath the phantom surface. Dose results of 4.4 cGy, 3.5 cGy and 2.0 cGy were measured with the films situated at depths of 6.5 cm, 9.5 cm and 14.5 cm, respectively. An average dose of 4.2 cGy to the fetus was derived from the above values. A corresponding dose of 3.2 cGy was also calculated based on results obtained using EBT3 films situated upon the patient skin. ConclusionsThe measured fetal doses are below the threshold of 10 cGy for congenital malformations, mental and growth retardation effects. The radiogenic cancer risk to the live-born embryo was estimated less than 0.3% over the normal incidence. The treatment was administered successfully, allowing the patient to deliver a healthy child.

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

Protective potential of α-tocopherol supplementation against ethanol-induced dysmorphogenesis in postimplantation murine embryos

Abstract Objective To assess the protective potential of α-tocopherol on ethanol-induced dysmorphogenesis in 10.5 embryonic day (ED) mouse embryos. Methods Forty female mice were randomly assigned into control (CON), positive control (ETOH), low-, medium and high-α-tocopherol-supplemented-Ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol, ETOH, LTOC, MTOC and HTOC groups received 20% ethanol in drinking water. The supplemented groups were given respective dosages of α-tocopherol, 0.410, 0.819 and 1.640 mg/g body weight, at 14 days before mating until the 9th day of gestation. The 10.5 ED embryos were assessed for embryo weight, head- and crown-rump length, and morphological scoring of brain and sensory vesicles, flexion and somites. The embryo yield was assessed by counting the number of full-term developed embryos from the bulging implantation sites while resorption was assessed by counting the bulging implantation sites but without formed embryos. Results The weight and head- and crown-rump length of the embryos from the α-tocopherol supplemented groups were comparable to the control. These were significantly higher than that of positive control ( P P P Conclusion The medium and high dosages of α-tocopherol exhibited a protective effect on ethanol-induced dysmorphogenesis.

Morphological development of eggs, larvae, and juvenile of Sebastes koreanus (Scorpaeniformes: Scorpaenidae) from the Yellow Sea

A total of 11 larvae [6.41–8.90 mm body length (BL)] and a juvenile (18.60 mm BL) of the genus Sebastes, collected from the Yellow Sea in 2012 and 2013, were identified as Sebastes koreanus on the basis of molecular mitochondrial DNA cytochrome oxidase subunit I (mtDNA COI) sequences (578 base pairs). Eggs and yolk-sac larvae of S. koreanus were also obtained from a gravid fish (168.60 mm standard length). The eggs of S. koreanus collected in this study were spherically shaped, and melanophores were completely absent on the head and body of embryos just prior to hatching. Melanophores were also absent in yolk-sac larvae, except on the upper margin of the abdominal region. However, melanophores were present on the head and abdominal region of pelagic larvae, and in a few larvae melanophores were present behind the nape (although these are considered to be individual variations). Three transverse stripes were present in juvenile, corresponding to the coloration observed in adults. The release season of S. koreanus larvae is known to be in winter, but in the present study larvae (6.41–8.90 mm BL) and a fully mature female were collected in May. In addition, a juvenile of S. koreanus (23.31 mm total length) was collected in July. On this basis, the release season of S. koreanus larvae is estimated to be in spring.

A Cell Surface Biotinylation Assay to Reveal Membrane-associated Neuronal Cues: Negr1 Regulates Dendritic Arborization

A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.

Ethanol-induced impairment of polyamine homeostasis – A potential cause of neural tube defect and intrauterine growth restriction in fetal alcohol syndrome

IntroductionPolyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. MethodsCD-1 mice received two intraperitoneal injections of 3g/kg ethanol at 4h intervals 8.75days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5dpc and polyamine concentrations were determined. ResultsNo measurable quantity of polyamines could be detected in the embryo head at 9.5dpc, 12h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. DiscussionOur results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.

Descriptions of eggs of snailfishes (family Liparidae) from the Bering Sea and eastern North Pacific Ocean

Eggs of liparid fishes were collected by bottom trawl in the southeastern Bering Sea and removed from an oceanographic mooring cable off the coast of British Columbia, Canada. Eggs were large with embryos most commonly in late flexion to postflexion stages. Based on meristic counts and collection localities, eggs were identified as Elassodiscus tremebundus, Paraliparis rosaceus, and Careproctus sp. (either Careproctus colletti or Careproctus melanurus). Eggs of E. tremebundus are oval with a smooth, opaque chorion. Eggs of P. rosaceus are spherical and the chorion is sculptured with a uniformly pebbled texture. Eggs of Careproctus sp. are slightly oval and the chorion is smooth and opaque. Embryos of all species are unpigmented. A pelvic disk is present in embryos of E. tremebundus, but it is rudimentary: a small patch of tissue that was visible only after embryos were cleared and stained. Adults of the genus Paraliparis do not have a pelvic disk and no disk was observed in developing embryos. Embryos of Careproctus sp. have a large disk with rays visible in later stages. Hatching glands are present on the head and nape of embryos of all three species. Hatching glands of E. tremebundus are separated into bands across the snout, along the jaws and opercle, and in a patch on the nape, while those of P. rosaceus and Careproctus sp. are evenly distributed over the head and nape. This is the first full description of the embryonic stages of these taxa.

Skeletal gene expression in the temporal region of the reptilian embryos: implications for the evolution of reptilian skull morphology

Reptiles have achieved highly diverse morphological and physiological traits that allow them to exploit various ecological niches and resources. Morphology of the temporal region of the reptilian skull is highly diverse and historically it has been treated as an important character for classifying reptiles and has helped us understand the ecology and physiology of each species. However, the developmental mechanism that generates diversity of reptilian skull morphology is poorly understood. We reveal a potential developmental basis that generates morphological diversity in the temporal region of the reptilian skull by performing a comparative analysis of gene expression in the embryos of reptile species with different skull morphology. By investigating genes known to regulate early osteoblast development, we find dorsoventrally broadened unique expression of the early osteoblast marker, Runx2, in the temporal region of the head of turtle embryos that do not form temporal fenestrae. We also observe that Msx2 is also uniquely expressed in the mesenchymal cells distributed at the temporal region of the head of turtle embryos. Furthermore, through comparison of gene expression pattern in the embryos of turtle, crocodile, and snake species, we find a possible correlation between the spatial patterns of Runx2 and Msx2 expression in cranial mesenchymal cells and skull morphology of each reptilian lineage. Regulatory modifications of Runx2 and Msx2 expression in osteogenic mesenchymal precursor cells are likely involved in generating morphological diversity in the temporal region of the reptilian skull.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-336) contains supplementary material, which is available to authorized users.

Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency by Modulating p53 Activity in Models of Diamond Blackfan Anemia

Abstract 512Ribosomal protein mutations are common in patients with Diamond Blackfan anemia (DBA), who have red cell aplasia and craniofacial abnormalities, and rps14 deficiency has been linked to 5q- myelodysplastic syndrome. We have characterized a zebrafish mutant in rps29, a ribosomal protein in the small subunit. Rps29−/− embryos have hematopoietic and endothelial defects, including decreased hematopoietic stem cells, defects in hemoglobinization, and decreased staining of vessel markers. Heads of mutant embryos are also morphologically affected, with increased apoptosis. Consistent with other models of DBA, knockdown of p53 completely rescues the rps29 hematopoietic and apoptotic phenotypes. We wanted to identify chemicals that could rescue the rps29 mutant phenotype. Using 600 compounds from known bioactive libraries, we performed an in vivo chemical screen for rescue of the rps29 apoptotic and endothelial defects. Treatment with one compound, A-3, improved the morphology of the apoptotic embryo head. A-3 is an inhibitor of calmodulin, which interacts with a host of calmodulin-dependent enzymes and can bind to other proteins in the cell, including p21. W-7, another calmodulin inhibitor related to A-3, can rescue the endothelial defect in the rps29 mutant. Calmodulin inhibition in rps29 mutant embryos rescues the hemoglobin defect. Furthermore, structurally unrelated chemicals that inhibit calmodulin rescue the endothelial defect. We hypothesized that calmodulin inhibition was affecting some part of the p53 pathway. We tested whether A-3 had an effect on the phenotype in vitro in a cancer cell line where shRNA knockdown of RPS19 has been shown to stabilize p53 and induce p21. When rps19 is knocked down in A549 cells, treatment with calmodulin inhibitors resulted in a decrease of p21 protein, as measured by flow cytometry, although p53 protein levels were stable. This effect was also observed in primary human cord blood-derived CD34+ hematopoietic stem and progenitor cells. In situ immunofluorescence in A549s further revealed that nuclear localization of p53 and p21 upon RPS19 knockdown was disrupted by chemical treatment. Calmodulin inhibitors also rescue ribosomal protein deficiency-mediated erythroid defects both in vitro and in vivo. When RPS19 is knocked down in CD34+ cells, there is a defect in erythroid differentiation. Treatment with calmodulin inhibitors rescued production of CD71+ cells upon differentiation. In conclusion, we have demonstrated the use of calmodulin inhibitors as a novel approach for mediating p53 activation upon ribosomal protein knockdown, thereby rescuing the erythroid defects of ribosomal deficiency. Our data suggests that small molecules that inhibit calmodulin may be effective therapies for patients with ribosomal deficiency. Disclosures:Ebert:Celgene: Consultancy; Genoptix: Consultancy. Zon:Fate Therapeutics: Founder Other.

Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II–mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

Early morphological variation and induction of phenotypic plasticity in Patagonian pejerrey

The aim of this work was to study two aspects of phenotypic plasticity in the Patagonian pejerrey Odontesthes hatcheri (Teleostei: Atherinopsidae) the dependence of the early morphology on developmental time and temperature, and the induction of morphological changes by controlled feeding in juveniles. Newly hatched free embryos, incubated at two different temperatures (13 and 18oC), and juveniles were used for the study and induction of phenotypic plasticity. Body and head shapes were analyzed with geometric morphometrics and linear measurements. Our results showed that shape variation at hatching was related to the bending of the embryo head on the yolk sac, increasing the head-trunk angle due to progressive straightening of the embryo. The head-trunk angle was related with temperature at incubation, with embryos incubated at higher temperature being more bent. Embryos that hatched earlier had bigger yolk sacs than those that hatched later. In juveniles, controlled feeding experiments added new morphological variation to that of wild juveniles. In all comparisons, the slenderness of the head, the size of premaxilla and jaw, and the position of the eye showed an enlarged variation due to controlled feeding. These results will contribute to comprehending the complexity of the morphological variation of O. hatcheri.

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.

Coordinate development of skin cells and cutaneous sensory axons in zebrafish

Peripheral sensory axons innervate the epidermis early in embryogenesis to detect touch stimuli. To characterize the time course of cutaneous innervation and the nature of interactions between sensory axons and skin cells at early developmental stages, we conducted a detailed analysis of cutaneous innervation in the head, trunk, and tail of zebrafish embryos and larvae from 18 to 78 hours postfertilization. This analysis combined live imaging of fish expressing transgenes that highlight sensory neurons and skin cells, transmission electron microscopy (TEM), and serial scanning electron microscopy (sSEM). In zebrafish, the skin initially consists of two epithelial layers, and all of the axons in the first wave of innervation are free endings. Maturation of the epithelium coincides with, but does not depend on, its innervation by peripheral sensory axons. We found that peripheral axons initially arborize between the two epithelial skin layers, but not within the basal lamina, as occurs in other organisms. Strikingly, as development proceeds, axons become tightly enveloped within basal keratinocytes, an arrangement suggesting that keratinocytes may serve structural or functional roles, akin to Schwann cells, in somatosensation mediated by these sensory neurons.

Automated analysis of zebrafish images for phenotypic changes in drug discovery

Zebrafish has become one of the most popular and useful models in cell biology, development, and drug discovery. Because zebrafish embryo is transparent and can be observed under microscope without fixation, it is increasingly used in high-throughput screening. The small size of zebrafish embryos allows users to image them in a 96- or 384-well plate under various conditions, in turn, generating such a large amount of images that only automated analysis is feasible for processing and analyzing. We focus on developing an image processing algorithm to automatically quantify gene expression on zebrafish embryos that have been treated by various compounds. The challenge in this type of application includes aligning embryos of different orientations and automatically creating regions of interest (ROIs) to enclose specific areas in the head and torso of embryos. The image processing pipeline consists of alignment, segmentation, creation and quantification of ROIs. We test the algorithm using high-throughput images of zebrafish embryos obtained from an experiment screening compounds that may affect γ-secretase in Alzheimer's disease and results show that automated analysis can achieve satisfactory performance in a much shorter amount of time with a high level of objectivity.

Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

BackgroundThe tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.MethodsWild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.ResultsApoptosis-related genes (bad, bax, and bcl2), and cell cycle-related genes (cdk2, pcna, p27, and p57) showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.ConclusionOur study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos.

Novel Cleft Susceptibility Genes in Chromosome 6q

Cleft lip/palate is a defect of craniofacial development. In previous reports, chromosome 6q has been suggested as a candidate region for cleft lip/palate. A multipoint posterior probability of linkage analysis of multiplex families from the Philippines attributed an 88% probability of harboring a cleft-susceptibility gene to a narrower region on bands 6q14.2-14.3. We genotyped 2732 individuals from families and unrelated individuals with and without clefts to investigate the existence of possible cleft-susceptibility genes in this region. We found association of PRSS35 and SNAP91 genes with cleft lip/palate in the case-control cohort and in Caucasian families. Haplotype analyses support the individual associations with PRSS35. We found Prss35 expression in the head and palate of mouse embryos at critical stages for palatogenesis, whereas Snap91 was expressed in the adult brain. We provide further evidence of the involvement of chromosome 6q in cleft lip/palate and suggest PRSS35 as a novel candidate gene.