Prechordal structures act cooperatively in early trabeculae development of gnathostome skull.

The vertebrate skull is formed by mesoderm and neural crest (NC) cells. The mesoderm contributes to the skull chordal domain, with the notochord playing an essential role in this process. The NC contributes to the skull prechordal domain, prompting investigation into the embryonic structures involved in prechordal neurocranium cartilage formation. The trabeculae cartilage, a structure of the prechordal neurocranium, arises at the convergence of prechordal plate (PCP), ventral midline (VM) cells of the diencephalon, and dorsal oral ectoderm. This study examines the molecular participation of these embryonic structures in gnathostome trabeculae development. PCP-secreted SHH induces its expression in VM cells of the diencephalon, initiating a positive feedback loop involving SIX3 and GLI1. SHH secreted by the VM cells of the diencephalon acts on the dorsal oral ectoderm, stimulating condensation of NC cells to form trabeculae. SHH from the prechordal region affects the expression of SOX9 in NC cells. BMP7 and SHH secreted by PCP induce NKX2.1 expression in VM cells of the diencephalon, but this does not impact trabeculae formation. Molecular cooperation between PCP, VM cells of the diencephalon, and dorsal oral ectoderm is crucial for craniofacial development by NC cells in the prechordal domain.

"Sitting-up vertigo as an expression of posterior semicircular canal heavy cupula and posterior semicircular canal short arm canalolithiasis".

Background: Vestibular symptoms on sitting-up are frequent on patients seen by vestibular specialists. Recently, a benign paroxysmal positional vertigo (BPPV) variant which elicits vestibular symptoms with oculomotor evidence of posterior semicircular canal (P-SCC) cupula stimulation on sitting-up was described and named sitting-up vertigo BPPV. A periampullar restricted P-SCC canalolithiasis was proposed as a causal mechanism. Objective: To describe new mechanisms of action for the sitting-up vertigo BPPV variant. Methods: Eighteen patients with sitting-up vertigo BPPV were examined with a pre-established set of positional maneuvers and follow-up until they resolved their symptoms and clinical findings. Results: All patients showed up-beating torsional nystagmus (UBTN) and vestibular symptoms on coming up from either Dix-Hallpike (DHM) or straight head-hanging maneuver. Sixteen out of 18 patients presented a sustained UBTN with an ipsitorsional component to the tested side on half-Hallpike maneuver (HH). A slower persistent contratorsional down-beating nystagmus was found in eleven out 18 patients tested on nose down position (ND). Conclusions: Persistent direction changing positional nystagmus on HH and ND positions indicative of P-SCC heavy cupula was found in 11 patients. A sustained UBTN on HH with the absence of findings on ND, which is suggestive of the presence of P-SCC short arm canalolithiasis, was found on 5 patients. All patients were treated with canalith repositioning maneuvers without success, but they resolved their findings by means of Brandt-Daroff exercises. We propose P-SCC heavy cupula and P-SCC short arm canalolithiasis as two new putative mechanisms for the sitting-up vertigo BPPV variant.

YAP1 Is a Potential Predictive Molecular Biomarker for Response to SMO Inhibitor in Medulloblastoma Cells.

Advances in genomics have led to the identification of twelve relevant molecular subtypes within medulloblastoma (MB). The alpha subtype of Sonic hedgehog-driven MB is resistant to therapy (including smoothened inhibitors) due to activation of genes from the non-canonical SHH pathway, such as MYCN, YAP1, or TP53. Using retrospective cohort microarray data, we found that YAP1 is overexpressed in SHH alpha MB and patients profiled as resistant to SMO inhibitors compared to good responders. Here, we performed YAP1 depletion via CRISPR/Cas9 in two in vitro models of SHH-like MB cells and found that this protein is involved in responsiveness to the SMO inhibitor regarding proliferation, apoptosis, and colony formation. Further, considering the synergic combination of YAP1 depletion with SMO inhibition, we assessed single-cell RNA-seq data from five patients and found that SMO and YAP1 are enriched within cells of SHH MB. Importantly, our data suggest that YAP1 is not only a reliable biomarker for cellular response to SMOi but may indicate prospective testing of combination therapy using YAP1 and SMO inhibitors in preclinical models of SHH MB.

New SHH and Known SIX3 Variants in a Series of Latin American Patients with Holoprosencephaly.

Holoprosencephaly (HPE) is the failure of the embryonic forebrain to develop into 2 hemispheres promoting midline cerebral and facial defects. The wide phenotypic variability and causal heterogeneity make genetic counseling difficult. Heterozygous variants with incomplete penetrance and variable expressivity in the SHH, SIX3, ZIC2, and TGIF1 genes explain ∼25% of the known causes of nonchromosomal HPE. We studied these 4 genes and clinically described 27 Latin American families presenting with nonchromosomal HPE. Three new SHH variants and a third known SIX3 likely pathogenic variant found by Sanger sequencing explained 15% of our cases. Genotype-phenotype correlation in these 4 families and published families with identical or similar driver gene, mutated domain, conservation of residue in other species, and the type of variant explain the pathogenicity but not the phenotypic variability. Nine patients, including 2 with SHH pathogenic variants, presented benign variants of the SHH, SIX3, ZIC2, and TGIF1 genes with potential alteration of splicing, a causal proposition in need of further studies. Finding more families with the same SIX3 variant may allow further identification of genetic or environmental modifiers explaining its variable phenotypic expression.

Testosterone exposure in prenatal life disrupts epithelial nuclear morphology, smooth muscle layer pattern, and FGF10 and Shh expression in prostate.

The aim of this study was to evaluate whether high levels of exogenous testosterone (T) interfere in prostate morphogenesis. Pregnant females were exposed to subcutaneous injections of T cypionate (500 µg/animal) at gestational days 20 and 22. Male and female pups were euthanized at postnatal days 1 and 15. 15-day-old males had only fibroblast growth factor 10 (FGF10) immunostaining and nuclear form factor altered by the treatment, whereas treated females (T1 and T15) had almost all analyzed parameters changed. T1 females showed an increased anogenital distance (AGD), whereas T15 females had both AGD and ovary weight increased. T1 females had a higher number of epithelial buds emerging from the urethral and vaginal epithelium. We observed ectopic prostatic tissue surrounding the vagina in both T1 and T15 females. Moreover, the ectopic acini of T15 females showed delayed luminal formation, and there was a thickening of the periacinar smooth muscle layer (SML). Finally, FGF10 immunostaining intensity decreased in both T15 male and female prostates. Indeed, Sonic hedgehog (Shh) was upregulated in T15 female prostates, whereas no difference was observed between the male groups. These data showed that exogenous T changed the nuclear morphology of prostate epithelial cells in both males and females. Surprisingly, smooth muscle hyperplasia was also observed in the ectopic female prostate. Moreover, T downregulated FGF10 in both male and female prostates. Interestingly, the results suggest that FGF10 downregulation is mediated by the upregulation of Shh in females. In conclusion, exogenous T disrupts prostate development, particularly, affecting, the female.

Bridging the treatment gap in infant medulloblastoma: molecularly informed outcomes of a globally feasible regimen.

BACKGROUND: Infant medulloblastoma represents an enormous challenge in neuro-oncology, due to their simultaneous high-risk of recurrence and high risk of severe neurodevelopmental sequelae with craniospinal irradiation. Currently infant medulloblastoma are treated with intensified protocols, either comprising intraventricular methotrexate or autologous transplant, both of which carry significant morbidity and are not feasible in the majority of the world. We sought to evaluate the molecular predictors of outcome in a cohort of infants homogeneously treated with induction chemotherapy, focal radiation and maintenance chemotherapy. METHODS: In a retrospective analysis, 29 young children treated with a craniospinal irradiation sparing strategy from Hospital Garrahan in Buenos Aires were profiled using Illumina HumanMethylationEPIC arrays, and correlated with survival. RESULTS: Twenty-nine children (range, 0.3-4.6 y) were identified, comprising 17 sonic hedgehog (SHH), 10 Group 3/4, and 2 non-medulloblastomas. Progression-free survival (PFS) across the entire cohort was 0.704 (95% CI: 0.551-0.899). Analysis by t-distributed stochastic neighbor embedding revealed 3 predominant groups, SHHß, SHHγ, and Group 3. Survival by subtype was highly prognostic with SHHγ having an excellent 5-year PFS of 100% (95% CI: 0.633-1) and SHHß having a PFS of 0.56 (95% CI: 0.42-1). Group 3 had a PFS of 0.50 (95% CI: 0.25-1). Assessment of neurocognitive outcome was performed in 11 patients; the majority of survivors fell within the low average to mild intellectual disability, with a median IQ of 73.5. CONCLUSIONS: We report a globally feasible and effective strategy avoiding craniospinal radiation in the treatment of infant medulloblastoma, including a robust molecular correlation along with neurocognitive outcomes.

Interplay between the RNA binding-protein Musashi and developmental signaling pathways.

Musashi comprises an evolutionarily conserved family of RNA-binding proteins (RBP) that regulate cell fate decisions during embryonic development and play key roles in the maintenance of self-renewal and differentiation of stem cells and adult tissues. More recently, several studies have shown that any dysregulation of MSI1 and MSI2 can lead to cellular dysfunctions promoting tissue instability and tumorigenesis. Moreover, several reports have characterized many molecular interactions between members of the Musashi family with ligands and receptors of the signaling pathways responsible for controlling normal embryonic development: Notch, Transforming Growth Factor Beta (TGF-ß), Wingless (Wnt) and Hedgehog Signaling (Hh); all of which, when altered, are strongly associated with cancer onset and progression, especially in pediatric tumors. In this context, the present review aims to compile possible cross-talks between Musashi proteins and members of the above cited molecular pathways for which dysregulation plays important roles during carcinogenesis and may be modulated by these RBP.

Stability and plasticity of positional memory during limb regeneration in Ambystoma mexicanum.

BACKGROUND: Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers. RESULTS: In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. CONCLUSIONS: Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.

Lupeol inhibits LPS-induced neuroinflammation in cerebellar cultures and induces neuroprotection associated to the modulation of astrocyte response and expression of neurotrophic and inflammatory factors.

In the central nervous system (CNS), neuroinflammation, especially that modulated by the cell response of astrocytes and microglia, is associated with damage to neurons in neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and, Multiple Sclerosis. Lupeol is a dietary triterpene that has demonstrated biological activities as antioxidant. This study investigated the anti-inflammatory and neuroprotective effects of lupeol in an in vitro model of neuroinflammation in primary cerebellar cultures. Cultures were obtained from 6-day-old Wistar rats, subjected to inflammatory damage with lipopolysaccharide (LPS, 1 µg/mL) and treated with lupeol (0.1 µM). We observed, after a 48-hour treatment, through Fluorjade-B staining and immunocytochemistry (ICQ) for ßIII-tubulin, that lupeol induced neuroprotection in cultures submitted to inflammatory damage. On the other hand, through ICQ for GFAP, it was possible to observe that lupeol modulated the astrocyte morphology for Bergmann glia-like phenotype and, especially for velate astrocyte-like phenotype, both phenotypes associated with the neuroprotective profile. Moreover, RT-qPCR analysis showed that lupeol induced the down-regulation of the mRNA expression for proinflammatory markers TNF, iNOS and NLRP3, as well as the production of nitric oxide (method of Greiss), which were up-regulated by LPS, and also induced up-regulation of the mRNA expression for arginase and IL-6 mRNA. In addition, lupeol induced up-regulation of mRNA expression for neurotrophins GDNF and NGF and also for the sonic hedgehog-Gli pathway. Together, these results lead to the conclusion that lupeol inhibits neuroinflammation in cerebellar cultures and induces neuroprotection associated with the modulation of astrocyte response and expression of neurotrophic and inflammatory factors.

Spatial and Quantitative Detection of BMP Activity in Mouse Embryonic Limb Buds.

Modulation of bone morphogenetic protein (BMP) activity is essential to the progression of limb development in the mouse embryo. Genetic disruption of BMP signaling at various stages of limb development causes defects ranging from complete limb agenesis to oligodactyly, polydactyly, webbing, and chondrodysplasia. To probe the state of BMP signaling in early limb buds, we designed two sets of primers to measure both spatially and quantitatively the transcription of nine key genes indicative of canonical BMP activity. One set is used to generate digoxigenin (DIG)-labeled antisense RNA probes for whole-mount mRNA in situ hybridization, while the second set is used for SYBR® Green-based quantitative PCR on limb bud cDNA. Here we describe step-by-step protocols for both methods around this specific set of genes.

Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus.

The phenotype of lens-ablated Mexican tetra (Astyanax mexicanus) compared to wild-type surface fish has been described and includes, among other effects, eye degeneration, changes in tooth number and cranial bone changes. Here, we investigate the spatiotemporal expression patterns of several key genes involved in the development of these structures. Specifically, we show that the expression of pitx2, bmp4 and shh is altered in the eye, oral jaw, nasal pit and forebrain in these lens-ablated fish. Furthermore, for the first time, we show altered pitx2 expression in the cavefish, which also has altered eye and tooth phenotypes. We thus provide evidence for a genetic linkage between the eye and tooth modules in this fish species. Furthermore, the altered pitx2 expression pattern, together with the described morphological features of the lens-ablated fish suggests that Astyanax mexicanus could be considered as an alternative teleost model organism in which to study Axenfeld-Rieger syndrome (ARS), a rare autosomal dominant developmental disorder that is associated with PITX2 and which has both ocular and non-ocular abnormalities.

Medulloblastoma, WNT-activated/SHH-activated: clinical impact of molecular analysis and histogenetic evaluation.

PURPOSE: Medulloblastoma (MDB) is a small cell poorly differentiated embryonal tumor of the cerebellum, which more frequently compromises children. Overall prognosis is favorable, but dependent of stage, histopathological pattern and molecular group. Approximately 30% of the affected patients will die from the disease. WHO 2016 Classification of Tumors of the Central Nervous System (CNS) has been classified MDB into four principal groups: WNT-activated MDB, SHH-activated MDB, group 3 MDB, and group 4 MDB. WNT-activated MDB is associated to monosomy 6, CTNNB1, DDX3X and TP53 mutations, beta-catenin nuclear immunoexpression, and a better prognosis than SHH-activated MDB. DISCUSSION: WNT-activated tumors account approximately for 10% of cases of MDBs, and are thought to arise from cells in the dorsal brain stem/lower rhombic lip progenitor cells. SHH-activated MDB more frequently arises in the lateral hemispheres of the cerebellum, and clinical outcome in this group is variable. TP53-mutant SHHactivated MDB usually shows the large cell/anaplastic pattern, and can be related to MYCN amplification, GLI2 amplification and 17p loss. TP53-wildtype SHH-activated MDB is more commonly of desmoplastic/nodular morphology, and can be related to PTCH1 deletion and 10q loss. Gene expression and methylation profiling is the gold standard for defining molecular groups of MDB. In immunohistochemistry assays, anti-GAB1 antibody expression is positive in tumors showing SHH pathway activation or PTCH mutation, while positive immunoexpression for YAP1 antibody can be only found in WNT-activated and SHH-activated MDB.

A highlight on Sonic hedgehog pathway.

Hedgehog (Hh) signaling pathway plays an essential role during vertebrate embryonic development and tumorigenesis. It is already known that Sonic hedgehog (Shh) pathway is important for the evolution of radio and chemo-resistance of several types of tumors. Most of the brain tumors are resistant to chemotherapeutic drugs, consequently, they have a poor prognosis. So, a better knowledge of the Shh pathway opens an opportunity for targeted therapies against brain tumors considering a multi-factorial molecular overview. Therefore, emerging studies are being conducted in order to find new inhibitors for Shh signaling pathway, which could be safely used in clinical trials. Shh can signal through a canonical and non-canonical way, and it also has important points of interaction with other pathways during brain tumorigenesis. So, a better knowledge of Shh signaling pathway opens an avenue of possibilities for the treatment of not only for brain tumors but also for other types of cancers. In this review, we will also highlight some clinical trials that use the Shh pathway as a target for treating brain cancer.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

Inhibition of the 3-hydroxy-3-methyl-glutaryl-CoA reductase induces orofacial defects in zebrafish.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects, which include a range of disorders with a complex etiology affecting formation of craniofacial structures. Some forms of syndromic OFCs are produced by defects in the cholesterol pathway. The principal enzyme of the cholesterol pathway is the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Our aim is to study whether defects of HMGCR function would produce orofacial malformation similar to those found in disorders of cholesterol synthesis. METHODS: We used zebrafish hmgcrb mutants and HMGCR inhibition assay using atorvastatin during early and late stages of orofacial morphogenesis in zebrafish. To describe craniofacial phenotypes, we stained cartilage and bone and performed in situ hybridization using known craniofacial markers. Also, we visualized neural crest cell migration in a transgenic fish. RESULTS: Our results showed that mutants displayed loss of cartilage and diminished orofacial outgrowth, and in some cases palatal cleft. Late treatments with statin show a similar phenotype. Affected-siblings displayed a moderate phenotype, whereas early-treated embryos had a minor cleft. We found reduced expression of the downstream component of Sonic Hedgehog-signaling gli1 in ventral brain, oral ectoderm, and pharyngeal endoderm in mutants and in late atorvastatin-treated embryos. CONCLUSION: Our results suggest that HMGCR loss-of-function primarily affects postmigratory cranial neural crest cells through abnormal Sonic Hedgehog signaling, probably induced by reduction in metabolites of the cholesterol pathway. Malformation severity correlates with the grade of HMGCR inhibition, developmental stage of its disruption, and probably with availability of maternal lipids. Together, our results might help to understand the spectrum of orofacial phenotypes found in cholesterol synthesis disorders. Birth Defects Research (Part A) 106:814-830, 2016. © 2016 Wiley Periodicals, Inc.

Morphology of the eyeball, orbit and retina of Atlantic salmon (Salmo salar) alevins under hypoxic conditions / Morfología del bulbo ocular, órbita y retina de alevines del salmón del Atlántico (Salmo salar) bajo condiciones hipóxicas

It has been demonstrated that hypoxia retards the growth of fish, reduces the survival of their larvae, deforms their vertebral column, but despite this teleost fish have the ability to completely regenerate many of their tissues, particularly the retina. As we do not have enough information about the effects of hypoxia on the eyeball, orbit and retina of Atlantic salmon (Salmo salar), we propose the following objectives: 1) Compare the morphological changes of the eyeball of fish subject to hypoxia and normoxia. 2) Determine changes in the orbit structure. 3) Describe the retina of salmon alevins. 4). Recognize hypoxic cells using the anti-Hif1a antibody in the retina of alevins as a sensor. 5) Determine the Shh morphogenic expression in alevins exposed to different times of hypoxia. Around 1,000 Salmo salar alevins were placed in a continuous water flow of 9 °C at 100 % SatO2 and alevins maintained at a hypoxia of 60 % SatO2. The latter were transferred to normoxia (at days two, four, and eight after hatching). A control group maintained at continuous normoxia and another at continuous hypoxia was also considered. All the alevins were euthanized at 950 UTAs (±2 months after hatching). Diaphonization (double-stain) according to the Hanken & Wassersug technique was undertaken to describe the morphology of the periocular cartilage and to measure the ocular diameter. The HIF-1a factor antibody 1:50, and the anti-Shh antibody dilution of 1:100 were used. The alevins after hatching had large eyeballs with the optic cup having an embryonic shape, even a choroidal fissure. The greatest thickness was observed in the nasal ventral zone which corresponds to a zone of pluripotent cells. The optic cup aspect with embryonic characteristics has only been reported in salmonids. The central retina of the alevins those were cultivated with a 60 % saturation of O2 for two, four or eight days had positive immunostaining when analyzed with the anti-HIF1a antibody hypoxia sensor. The inner ganglion and nuclear layers had immunopositive cells, with the highest in the alevins that were two days in hypoxia and the lowest when the hypoxia was chronic. Nevertheless, in the latter case the alevins had anatomical deformation of the eyeball and periocular cartilage. The anti-Shh antibody clearly shows a gradient that is expressed in the germinative zone and in the cells of the inner ganglion and nuclear layers. The eyeball and particularly the retina in salmon alevins are an example of neuronal plasticity and neurogenesis.

Se ha demostrado que la hipoxia retarda el crecimiento de los peces, reduce la supervivencia de sus larvas, deforma su columna vertebral, pero a pesar de esto, este pez teleósteo tiene la capacidad de regenerar completamente muchos de sus tejidos, en particular la retina. Como no existe suficiente información sobre los efectos de la hipoxia en el bulbo ocular, la órbita y retina del salmón del Atlántico (Salmo salar), los objetivos de este trabajo fueron: 1) Comparar los cambios morfológicos del bulbo ocular del pescado sujetos a hipoxia y normoxia; 2) Determinar los cambios en la estructura de la órbita; 3) Describir la retina de los alevines de salmón; 4) Reconocer las células hipóxicas utilizando el anticuerpo anti-Hif1a en la retina de alevines como un sensor; 5) Determinar la expresión morfogenética de Shh en alevines expuestos a diferentes momentos de hipoxia. Alrededor de 1.000 alevines Salmo salar se colocaron en un flujo continuo de agua a 9 °C, con 100 % de SatO2 y otros alevines se mantuvieron con una hipoxia de 60 % SatO2. Estos últimos fueron trasladados a normoxia (en los días dos, cuatro y ocho después de la eclosión). Un grupo control se mantuvo a normoxia continua y otro grupo a hipoxia continua. Todos los alevines se sacrificaron a 950 UTA (+ dos meses después de la eclosión). Se realizcón una diafonización (doble tinción), de acuerdo con la técnica de Hanken & Wassersug, para describir la morfología del cartílago periocular y para medir el diámetro ocular. Se utilizaron el anticuerpo anti-Hif1a a una dilución 1:50, y el anticuerpo anti-Shh a una dilución de 1:100. Los alevines después de la eclosión presentaron grandes bulbos oculares, con la copa óptica con forma embrionaria, incluso una fisura coroidea. El mayor espesor se observó en la zona ventral nasal que corresponde a una zona de células pluripotentes. El aspecto de la copa óptica con características embrionarias sólo se ha informado en los salmónidos. La retina central de los alevines fueron cultivadas con una saturación de 60 % de O2 para dos, cuatro y ocho días, y presentó inmunotinción positiva cuando se analizó con el sensor de hipoxia, el anticuerpo anti-HIF1a. El ganglio interior y las capas nucleares presentaron células immunopositivas, con los niveles más altos en los alevines con dos días de hipoxia y niveles más bajos en hipoxia crónica. Sin embargo, en éste último caso los alevines presentaron una deformación anatómica del bulbo ocular y el cartílago periocular. El anticuerpo anti-Shh mostró claramente un gradiente expresado en la zona germinativa y en las células del ganglio interior y las capas nucleares. El bulbo ocular y en particular la retina en alevines de salmón son un ejemplo de plasticidad neuronal y neurogénesis.

Prenatal caffeine intake differently affects synaptic proteins during fetal brain development.

Caffeine is the psychostimulant most consumed worldwide. However, little is known about its effects during fetal brain development. In this study, adult female Wistar rats received caffeine in drinking water (0.1, 0.3 and 1.0 g/L) during the active cycle in weekdays, two weeks before mating and throughout pregnancy. Cerebral cortex and hippocampus from embryonic stages 18 or 20 (E18 or E20, respectively) were collected for immunodetection of the following synaptic proteins: brain-derived neurotrophic factor (BDNF), TrkB receptor, Sonic Hedgehog (Shh), Growth Associated Protein 43 (GAP-43) and Synaptosomal-associated Protein 25 (SNAP-25). Besides, the estimation of NeuN-stained nuclei (mature neurons) and non-neuronal nuclei was verified in both brain regions and embryonic periods. Caffeine (1.0 g/L) decreased the body weight of embryos at E20. Cortical BDNF at E18 was decreased by caffeine (1.0 g/L), while it increased at E20, with no major effects on TrkB receptors. In the hippocampus, caffeine decreased TrkB receptor only at E18, with no effects on BDNF. Moderate and high doses of caffeine promoted an increase in Shh in both brain regions at E18, and in the hippocampus at E20. Caffeine (0.3g/L) decreased GAP-43 only in the hippocampus at E18. The NeuN-stained nuclei increased in the cortex at E20 by lower dose and in the hippocampus at E18 by moderate dose. Our data revealed that caffeine transitorily affect synaptic proteins during fetal brain development. The increased number of NeuN-stained nuclei by prenatal caffeine suggests a possible acceleration of the telencephalon maturation. Although some modifications in the synaptic proteins were transient, our data suggest that caffeine even in lower doses may alter the fetal brain development.

A novel ZRS mutation leads to preaxial polydactyly type 2 in a heterozygous form and Werner mesomelic syndrome in a homozygous form.

Point mutations in the zone of polarizing activity regulatory sequence (ZRS) are known to cause human limb malformations. Although most mutations cause preaxial polydactyly (PPD), triphalangeal thumb (TPT) or both, a mutation in position 404 of the ZRS causes more severe Werner mesomelic syndrome (WMS) for which malformations include the distal arm or leg bones in addition to the hands and/or feet. Of more than 15 reported families with ZRS mutations, only one homozygous individual has been reported, with no change in phenotype compared with heterozygotes. Here, we describe a novel point mutation in the ZRS, 402C>T (AC007097.4:g.105548C>T), that is transmitted through two Mexican families with one homozygous individual. The homozygous phenotype for this mutation, WMS, is more severe than the numerous heterozygous individuals genotyped from both families who have TPT and PPD. A mouse transgenic enhancer assay shows that this mutation causes an expansion of the enhancer's expression domain in the developing mouse limb, confirming its pathogenicity. Combined, our results identify a novel ZRS mutation in the Mexican population, 402C>T, and suggest that a dosage effect exists for this ZRS mutation.

Molecular analysis of holoprosencephaly in South America.

Holoprosencephaly (HPE) is a spectrum of brain and facial malformations primarily reflecting genetic factors, such as chromosomal abnormalities and gene mutations. Here, we present a clinical and molecular analysis of 195 probands with HPE or microforms; approximately 72% of the patients were derived from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and 82% of the patients were newborns. Alobar HPE was the predominant brain defect in almost all facial defect categories, except for patients without oral cleft and median or lateral oral clefts. Ethmocephaly, cebocephaly, and premaxillary agenesis were primarily observed among female patients. Premaxillary agenesis occurred in six of the nine diabetic mothers. Recurrence of HPE or microform was approximately 19%. The frequency of microdeletions, detected using Multiplex Ligation-dependant Probe Amplification (MLPA) was 17% in patients with a normal karyotype. Cytogenetics or QF-PCR analyses revealed chromosomal anomalies in 27% of the probands. Mutational analyses in genes SHH, ZIC2, SIX3 and TGIF were performed in 119 patients, revealing eight mutations in SHH, two mutations in SIX3 and two mutations in ZIC2. Thus, a detailed clinical description of new HPE cases with identified genetic anomalies might establish genotypic and phenotypic correlations and contribute to the development of additional strategies for the analysis of new cases.