Disruption of planar cell polarity activity leads to developmental biliary defects

Planar cell polarity (PCP) establishes polarity within an epithelial sheet. Defects in PCP are associated with developmental defects involving directional cell growth, including defects in kidney tubule elongation that lead to formation of kidney cysts. Given the strong association between kidney cyst formation and developmental biliary defects in patients and in animal models, we investigated the importance of PCP in biliary development. Here we report that in zebrafish, morpholino antisense oligonucleotide-mediated knockdown of PCP genes including prickle-1a (pk1a) led to developmental biliary abnormalities, as well as localization defects of the liver and other digestive organs. The defects in biliary development appear to be mediated via downstream PCP targets such as Rho kinase, Jun kinase (JNK), and both actin and microtubule components of the cytoskeleton. Knockdown of pk1a led to decreased expression of vhnf1, a homeodomain gene previously shown to be involved in biliary development and in kidney cyst formation; forced expression of vhnf1 mRNA led to rescue of the pk1a morphant phenotype. Our results demonstrate that PCP plays an important role in vertebrate biliary development, interacting with other factors known to be involved in biliary morphogenesis.     

Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b^−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a^−/− zebrafish. The results showed that the expression of runx2b in bmp2a^−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.     

Carboxyl ester lipase is highly conserved in utilizing maternal supplied lipids during early development of zebrafish and human

Carboxyl ester lipase (Cel), is a lipolytic enzyme secreted by the pancreas, which hydrolyzes various species of lipids in the gut. Cel is also secreted by mammary gland during lactation and exists in breast milk. It facilitates dietary fat digestion and absorption, thus contributing to normal infant development. This study aimed to examine whether the Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants, and how Cel contributes to the utilization of yolk lipids in zebrafish. The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf, and in the exocrine pancreas after 72 hpf. The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention. The total cholesterol, cholesterol ester, free cholesterol, and triglyceride were reduced in the morphants' body while accumulated in the yolk (except triglyceride). The FFA content of whole embryos was much lower in morphants than in standard controls. Moreover, the delayed development in cel (cel1/cel2) double morphants was partially rescued by FFA and cholesterol supplementation. Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants. Correspondingly, shrunken midbrain tectum, microphthalmia, pigmentation-delayed eyes as well as down-regulated Shh target genes were observed in the CNS of double morphants. Interestingly, cholesterol injections reversed these CNS alterations. Our findings suggested that cel genes participate in the lipid releasing from yolk sac to developing body, thereby contributing to the normal growth rate and CNS development in zebrafish.     

Expression and Functional Characterization of Smyd1a in Myofibril Organization of Skeletal Muscles

Background

Smyd1, the founding member of the Smyd family including Smyd-1, 2, 3, 4 and 5, is a SET and MYND domain containing protein that plays a key role in myofibril assembly in skeletal and cardiac muscles. Bioinformatic analysis revealed that zebrafish genome contains two highly related smyd1 genes, smyd1a and smyd1b. Although Smyd1b function is well characterized in skeletal and cardiac muscles, the function of Smyd1a is, however, unknown.

Methodology/Principal Findings

To investigate the function of Smyd1a in muscle development, we isolated smyd1a from zebrafish, and characterized its expression and function during muscle development via gene knockdown and transgenic expression approaches. The results showed that smyd1a was strongly expressed in skeletal muscles of zebrafish embryos. Functional analysis revealed that knockdown of smyd1a alone had no significant effect on myofibril assembly in zebrafish skeletal muscles. However, knockdown of smyd1a and smyd1b together resulted in a complete disruption of myofibril organization in skeletal muscles, a phenotype stronger than knockdown of smyd1a or smyd1b alone. Moreover, ectopic expression of zebrafish smyd1a or mouse Smyd1 transgene could rescue the myofibril defects from the smyd1b knockdown in zebrafish embryos.

Conclusion/Significance

Collectively, these data indicate that Smyd1a and Smyd1b share similar biological activity in myofibril assembly in zebrafish embryos. However, Smyd1b appears to play a major role in this process.     

Neurologic and ocular phenotype in Pitt–Hopkins syndrome and a zebrafish model

In this study, we performed an in-depth analysis of the neurologic and ophthalmologic phenotype in a patient with Pitt–Hopkins syndrome (PTHS), a disorder characterized by severe mental and motor retardation, carrying a uniallelic TCF4 deletion, and studied a zebrafish model. The PTHS-patient was characterized by high-resolution magnetic resonance imaging (MRI) with diffusion tensor imaging to analyze the brain structurally, spectral-domain optical coherence tomography to visualize the retinal layers, and electroretinography to evaluate retinal function. A zebrafish model was generated by knockdown of tcf4-function by injection of morpholino antisense oligos into zebrafish embryos and the morphant phenotype was characterized for expression of neural differentiation genes neurog1, ascl1b, pax6a, zic1, atoh1a, atoh2b. Data from PTHS-patient and zebrafish morphants were compared. While a cerebral MRI-scan showed markedly delayed myelination and ventriculomegaly in the 1-year-old PTHS-patient, no structural cerebral anomalies including no white matter tract alterations were detected at 9 years of age. Structural ocular examinations showed highly myopic eyes and an increase in ocular length, while retinal layers were normal. Knockdown of tcf4-function in zebrafish embryos resulted in a developmental delay or defects in terminal differentiation of brain and eyes, small eyes with a relative increase in ocular length and an enlargement of the hindbrain ventricle. In summary, tcf4-knockdown in zebrafish embryos does not seem to affect early neural patterning and regionalization of the forebrain, but may be involved in later aspects of neurogenesis and differentiation. We provide evidence for a role of TCF4/E2-2 in ocular growth control in PTHS-patients and the zebrafish model.