Recurrent De Novo Mutations in PACS1 Cause Defective Cranial-Neural-Crest Migration and Define a Recognizable Intellectual-Disability Syndrome

We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.     

Isotretinoin teratogenicity in mouse whole embryo culture

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.     

FGF signals from the nasal pit are necessary for normal facial morphogenesis

Fibroblast growth factors (FGFs) are required for brain, pharyngeal arch, suture and neural crest cell development and mutations in the FGF receptors have been linked to human craniofacial malformations. To study the functions of FGF during facial morphogenesis we locally perturb FGF signalling in the avian facial prominences with FGFR antagonists, foil barriers and FGF2 protein. We tested 4 positions with antagonist-soaked beads but only one of these induced a facial defect. Embryos treated in the lateral frontonasal mass, adjacent to the nasal slit developed cleft beaks. The main mechanisms were a block in proliferation and an increase in apoptosis in those areas that were most dependent on FGF signaling. We inserted foil barriers with the goal of blocking diffusion of FGF ligands out of the lateral edge of the frontonasal mass. The barriers induced an upregulation of the FGF target gene, SPRY2 compared to the control side. Moreover, these changes in expression were associated with deletions of the lateral edge of the premaxillary bone. To determine whether we could replicate the effects of the foil by increasing FGF levels, beads soaked in FGF2 were placed into the lateral edge of the frontonasal mass. There was a significant increase in proliferation and an expansion of the frontonasal mass but the skeletal defects were minor and not the same as those produced by the foil. Instead it is more likely that the foil repressed FGF signaling perhaps mediated by the increase in SPRY2 expression. In summary, we have found that the nasal slit is a source of FGF signals and the function of FGF is to stimulate proliferation in the cranial frontonasal mass. The FGF independent regions correlate with those previously determined to be dependent on BMP signaling. We propose a new model whereby, FGF-dependent microenvironments exist in the cranial frontonasal mass and caudal maxillary prominence and these flank BMP-dependent regions. Coordination of the proliferation in these regions leads ultimately to normal facial morphogenesis.     

Local retinoid signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH

Correlations between facial anomalies and brain defects are well characterized throughout the clinical literature, yet a developmental basis for this association has not been identified. We demonstrate that the frontonasal process, which gives rise to the mid- and upper face, and the forebrain are linked early in their morphogenesis by a local retinoid signaling event that maintains the expression of key regulatory molecules. First, we show that aldehyde dehydrogenase 6, which synthesizes the ligand, retinoic acid, is localized to the ventral epithelium of the presumptive frontonasal process of chick embryos. At least two retinoid receptors are expressed in adjacent populations of mesenchyme. Second, using synthetic pan-specific retinoid antagonists, we transiently inhibit the ability of retinoid receptors to bind retinoic acid in the rostral head and we generate embryos with a hypoplastic forebrain, fused eyes, and no frontonasal process-derived structures such as the upper beak. These defects are not due to eliminating mesenchymal progenitors, as neural crest cells still migrate into the frontonasal process, despite disruptions to retinoid signaling. Rather, these malformations result from loss of fibroblast growth factor 8 and sonic hedgehog expression, which leads to increased programmed cell death and decreased proliferation in the forebrain and frontonasal process. Most significantly, we can rescue the morphological defects by re-introducing retinoic acid, or fibroblast growth factor and sonic hedgehog proteins into antagonist-treated embryos. We propose that the local source of retinoic acid in the rostral head initiates a regulatory cascade that coordinates forebrain and frontonasal process morphogenesis.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.     

Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

Background

Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.

Methodology

Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.

Conclusion

Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERα and ERβ. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.     

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.     

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant’s phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.     

Expression of zebrafish mab21 genes marks the differentiating eye, midbrain and neural tube

Mab21 homolog in Caenorhabditis elegans is expressed in the sensory ray cells and determines the ray identity (Dev. Dyn. 221 (2001) 422). Similarly, vertebrate homologs of this gene have been shown to express in the craniofacial tissues, limb bud and neural tissues. We report here the cloning and expression analysis of two zebrafish mab21 genes. Both genes have a conserved open reading frame of 1080 nucleotides. Phylogenetic analysis suggested that the two family subgroups existed prior to the divergence of vertebrates. Their expression profiles revealed that mab21l1 was turned on in embryos as early as 8 h post-fertilization (hpf) while mab21l2 was first detected at around 11 hpf. In situ hybridization results showed that expression of these mab21 genes marked the early differentiating olfactory bulbs, eye primordia, midbrain and subsequently the branchial pouches and neural tube.     

团头鲂MSTN基因cDNA结构、表达及过表达对胚胎发育的影响

研究通过cDNA末端快速扩增法(RACE)克隆得到团头鲂生长抑制素(MSTN)基因的cDNA全长并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果表明团头鲂MSTN基因的cDNA全长为2187 bp, ORF(开放阅读框)大小为1128 bp, 编码376个氨基酸。组织逆转录PCR (RT-PCR)结果显示, MSTN基因在肌肉、脑和精巢组织中大量表达, 肝脏、脾脏和卵巢组织中的少量表达, 肠、腮、心、眼和肾组织中的微量表达。胚胎逆转录PCR (RT-PCR)结果显示, 在0—44 hpf胚胎发育阶段, MSTN基因表达量较低; 而在48—52 hpf胚胎发育阶段, MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示, 胚胎发育的16 hpf时期MSTN基因主要在脊索中表达, 胚胎发育的28 hpf和55 hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示, 胚胎在体节发生期出现前-后轴拉长, 背-腹轴变短; 脊索发生扭曲, 强烈抑制体节发育而导致不分化等现象。研究为后续团头鲂MSTN基因的功能研究及团头鲂分子育种提供相关参考依据。     

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.     

The patterns on chondrogenesis of cells from facial primordia of chick embryos in micromass culture

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.