Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo as well as dosage, duration and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using human embryonic stem cells. Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells upon exposure to 100 mM ethanol for 48 h corresponding to the blood alcohol levels for binge drinkers. Whole-genome analysis revealed a spatiotemporal dysregulation of neuronal- and glial-specific gene expression that play critical roles in central nervous system (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated extracellular signal-regulated kinases signaling in alcohol-treated cells. Consistent with this finding, ingenuity pathway analysis and micro-RNA profiling demonstrated up-regulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of tubulin, beta 3 class III+ neurons was greatly reduced in response to alcohol. Finally, in vivo studies using zebrafish embryos reconfirmed the in vitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of extracellular signal-regulated kinases pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging.

Dysregulation of Wnt-Signaling and a Candidate Set of miRNAs Underlie the Effect of Metformin on Neural Crest Cell Development.

Neural crest cells (NCC) are a population of epithelial cells that arise from the dorsal tube and undergo epithelial-mesenchymal transition (EMT) eventually generating tissues from peripheral nervous system, melanocytes, craniofacial cartilage, and bone. The antidiabetic drug metformin reportedly inhibits EMT in physiological conditions like cancer and fibrosis. We hypothesize that perturbation of EMT may also contribute to developmental disabilities associated with neural crest (NC) development. To understand the molecular network underlying metformin action during NC formation, we first differentiated murine embryonic stem (ES) cells into NCC and characterized them by demonstrating spatiotemporal regulation of key markers. Metformin treatment prompted a delay in delamination of NCC by inhibiting key markers like Sox-1, Sox-9, HNK-1, and p-75. We then revealed that metformin impedes Wnt axis, a major signaling pathway active during NC formation via DVL-3 inhibition and impairment in nuclear translocation of ß-catenin. Concomitantly we identified and tested a candidate set of miRNAs that play a crucial role in NC cell fate determination. Further studies involving loss and gain of function confirmed that NCC specifiers like Sox-1 and Sox-9 are direct targets of miR-200 and miR-145, respectively and that they are essentially modulated by metformin. Our in vitro findings were strongly supported by in vivo studies in zebrafish. Given that metformin is a widely used drug, for the first time we demonstrate that it can induce a delayed onset of developmental EMT during NC formation by interfering with canonical Wnt signaling and mysregulation of miR-145 and miR-200.

nlz1 is required for cilia formation in zebrafish embryogenesis.

The formation of cilia is a fundamental developmental process affecting diverse functions such as cellular signaling, tissue morphogenesis and body patterning. However, the mechanisms of ciliogenesis during vertebrate development are not fully understood. In this report we describe a novel role of the Nlz1 protein in ciliogenesis. We demonstrate morpholino-mediated knockdown of nlz1 in zebrafish causes abnormal specification of the cells of Kupffer's vesicle (KV); a severe reduction of the number of cilia in KV, the pronephros, and the neural floorplate; and a spectrum of later phenotypes reminiscent of human ciliopathies. In vitro and in vivo data indicate that Nlz1 acts downstream of Foxj1a and Wnt8a/presumed canonical Wnt signaling. Furthermore, Nlz1 contributes to motile cilia formation by positively regulating Wnt11/presumed non-canonical Wnt signaling. Together, our data suggest a novel role of nlz1 in ciliogenesis and the morphogenesis of multiple tissues.

Kctd15 inhibits neural crest formation by attenuating Wnt/beta-catenin signaling output.

Neural crest (NC) precursors are stem cells that are capable of forming many cell types after migration to different locations in the embryo. NC and placodes form at the neural plate border (NPB). The Wnt pathway is essential for specifying NC versus placodal identity in this cell population. Here we describe the BTB domain-containing protein Potassium channel tetramerization domain containing 15 (Kctd15) as a factor expressed in the NPB that efficiently inhibits NC induction in zebrafish and frog embryos. Whereas overexpression of Kctd15 inhibited NC formation, knockdown of Kctd15 led to expansion of the NC domain. Likewise, NC induction by Wnt3a plus Chordin in Xenopus animal explants was suppressed by Kctd15, but constitutively active beta-catenin reversed Kctd15-mediated suppression of NC induction. Suppression of NC induction by inhibition of Wnt8.1 was rescued by reduction of Kctd15 expression, linking Kctd15 action to the Wnt pathway. We propose that Kctd15 inhibits NC formation by attenuating the output of the canonical Wnt pathway, thereby restricting expansion of the NC domain beyond its normal range.

Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure.

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. Here, we profile global gene expression during optic fissure closure using laser capture microdissected (LCM) tissue from the margins of the fissure. From these data, we identify a unique role for the C(2)H(2) zinc finger proteins Nlz1 and Nlz2 in normal fissure closure. Gene knockdown of nlz1 and/or nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. We also identify misregulation of pax2 in the developing eye of morphant fish, suggesting that Nlz1 and Nlz2 act upstream of the Pax2 pathway in directing proper closure of the optic fissure.

Notch signaling regulates endocrine cell specification in the zebrafish anterior pituitary.

The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion.

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.

aldh7a1 regulates eye and limb development in zebrafish.

Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of aldh7a1 as a gene necessary for normal eye development. We show that morpholino knockdown of aldh7a1 in zebrafish causes uveal coloboma and misregulation of nlz1, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of aldh7a1 leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The aldh7a1 morphant phenotype is partially rescued by co-injection of nlz1 mRNA suggesting that nlz1 is functionally downstream of aldh7a1 in regulating cell proliferation in the optic cup. These results support a role of aldh7a1 in ocular development and skeletal abnormalities in zebrafish.

pitx3 defines an equivalence domain for lens and anterior pituitary placode.

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.