Paolo E. Forni

Neural crest and Ectodermal cells intermix in the nasal placode to give rise to GnRH-1 Neurons, Sensory Neurons and Olfactory Ensheathing Cells

The origin of GnRH-1 cells and olfactory ensheathing cells has been controversial. Genetic Cre-lox lineage tracing of the neural crest (NC) versus ectodermal contribution to the developing nasal placode was performed using two complementary mouse models, the NC-specific Wnt1Cre mouse line and an ectodermal-specific Crect mouse line. Using these lines we prove that the NC give rise to the olfactory ensheathing cells and subpopulations of GnRH-1 neurons, olfactory and vomeronasal cells. These data demonstrate that Schwann cells and olfactory ensheathing cells share a common developmental origin. Furthermore, the results indicate that certain conditions that impact olfaction and sexual development, such as Kallmann syndrome, may be in part neurocristopathies.

High Levels of Cre Expression in Neuronal Progenitors Cause Defects in Brain Development Leading to Microencephaly and Hydrocephaly

Hydrocephalus is a common and variegated pathology often emerging in newborn children after genotoxic insults during pregnancy (Hicks and D’Amato, 1980). Cre recombinase is known to have possible toxic effects that can compromise normal cell cycle and survival. Here we show, by using three independent nestin Cre transgenic lines, that high levels of Cre recombinase expression into the nucleus of neuronal progenitors can compromise normal brain development. The transgenics analyzed are the nestin Cre Balancer (Bal1) line, expressing the Cre recombinase with a nuclear localization signal, and two nestin CreERT2 (Cre recombinase fused with a truncated estrogen receptor) mice lines with different levels of expression of a hybrid CreERT2 recombinase that translocates into the nucleus after tamoxifen treatment. All homozygous Bal1 nestin Cre embryos displayed reduced neuronal proliferation, increased aneuploidy and cell death, as well as defects in ependymal lining and lamination of the cortex, leading to microencephaly and to a form of communicating hydrocephalus. An essentially overlapping phenotype was observed in the two nestin CreERT2 transgenic lines after tamoxifen mediated-CreERT2 translocation into the nucleus. Neither tamoxifen-treated wild-type nor nestin CreERT2 oil-treated control mice displayed these defects. These results indicate that some forms of hydrocephalus may derive from a defect in neuronal precursors proliferation. Furthermore, they underscore the potential risks for developmental studies of high levels of nuclear Cre in neurogenic cells.

Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development

Cerebellar development occurs mainly postnatally and implies cell proliferation and migration. Hepatocyte growth factor (HGF) and Met are involved in mediating these responses in other tissues and are coexpressed in the cerebellum. Here we show that Met is localized in granule cell precursors and that cultures of these cells respond to HGF with proliferation. To study the role of HGF and Met in the cerebellum in vivo, we produced a viable hypomorphic Met mutant by knocking in the met locus a point mutation to abrogate the receptor Grb2-binding site. A similar mutant was previously described as perinatal lethal. In this “first-generation” knock-in the recombinant locus retained the Neo cassette (Metgrb2/grb2neo+). In the knock-in presented here Neo was Loxed and excised by Cre recombinase, which led to higher tissue levels of Metgrb2 protein, sufficient to rescue viability. In Metgrb2/grb2neo− mice the size of the cerebellum was reduced and foliation defects were evident, especially in the central and posterior half of the vermis. Proliferation of granule precursors in vivo was 25% lower than in controls. In cultures of mutant granule cells HGF-induced microtubule-associated protein kinase activation was reduced and transient. Behavioral tests indicated a balance impairment in Metgrb2/grb2neo− mice. Altogether these data indicate that normal cerebellar development and, possibly, function, require HGF and Met, and that proliferation of granule cells in the cerebellum critically depends on full HGF/Met signaling.

BDNF, NT-3 and NGF induce distinct new Ca2+ channel synthesis in developing hippocampal neurons

Neurotrophins exert short‐ and long‐term effects on synaptic transmission. The mechanism underlying these forms of synaptic plasticity is unknown although it is likely that intracellular Ca2+ and presynaptic Ca2+ channels play a critical role. Here we show that BDNF, NGF and NT‐3 (10–100 ng/mL) exhibit a selective long‐term up‐regulation of voltage‐gated Ca2+ current densities in developing hippocampal neurons of 6–20 days in culture. NGF and NT‐3 appear more effective in up‐regulating L‐currents, while BDNF predominantly acts on non‐L‐currents (N, P/Q and R). The effects of the three neurotrophins were time‐ and dose‐dependent. The EC50 was comparable for BDNF, NGF and NT‐3 (10–16 ng/mL) while the time of half‐maximal activation was significantly longer for NGF compared to BDNF (58 vs. 25 h). Despite the increased Ca2+ current density, the neurotrophins did not alter the voltage‐dependence of channel activation, the kinetics parameters or the elementary properties of Ca2+ channels (single‐channel conductance, probability of opening and mean open time). Neurotrophin effects were completely abolished by coincubation with the nonspecific Trk‐receptor inhibitor K252a, the protein synthesis blocker anisomycin and the MAP‐kinase inhibitor PD98059, while cotreatment with the PLC‐γ blocker, U73122, was without effect. Immunocytochemistry and Western blotting revealed that neurotrophins induced an increased MAP‐kinase phosphorylation and its translocation to the nucleus. The present findings suggest that on a long time scale different neurotrophins can selectively up‐regulate different Ca2+ channels. The action is mediated by Trk‐receptors/MAP‐kinase pathways and induces an increased density of newly available Ca2+ channels with unaltered gating activity.

GnRH, anosmia and hypogonadotropic hypogonadism - Where are we?

Gonadotropin releasing hormone (GnRH) neurons originate the nasal placode and migrate into the brain during prenatal development. Once within the brain, these cells become integral components of the hypothalamic-pituitary-gonadal axis, essential for reproductive function. Disruption of this system causes hypogonadotropic hypogonadism (HH). HH associated with anosmia is clinically defined as Kallman syndrome (KS). Recent work examining the developing nasal region has shed new light on cellular composition, cell interactions and molecular cues responsible for the development of this system in different species. This review discusses some developmental aspects, animal models and current advancements in our understanding of pathologies affecting GnRH. In addition we discuss how development of neural crest derivatives such as the glia of the olfactory system and craniofacial structures control GnRH development and reproductive function.

Neural Crest and Olfactory System: New Prospective

Sensory neurons in vertebrates are derived from two embryonic transient cell sources: neural crest (NC) and ectodermal placodes. The placodes are thickenings of ectodermal tissue that are responsible for the formation of cranial ganglia as well as complex sensory organs that include the lens, inner ear, and olfactory epithelium. The NC cells have been indicated to arise at the edges of the neural plate/dorsal neural tube, from both the neural plate and the epidermis in response to reciprocal interactions Moury and Jacobson (Dev Biol 141:243–253, 1990). NC cells migrate throughout the organism and give rise to a multitude of cell types that include melanocytes, cartilage and connective tissue of the head, components of the cranial nerves, the dorsal root ganglia, and Schwann cells. The embryonic definition of these two transient populations and their relative contribution to the formation of sensory organs has been investigated and debated for several decades (Basch and Bronner-Fraser, Adv Exp Med Biol 589:24–31, 2006; Basch et al., Nature 441:218–222, 2006) review (Baker and Bronner-Fraser, Dev Biol 232:1–61, 2001). Historically, all placodes have been described as exclusively derived from non-neural ectodermal progenitors. Recent genetic fate-mapping studies suggested a NC contribution to the olfactory placodes (OP) as well as the otic (auditory) placodes in rodents (Murdoch and Roskams, J Neurosci Off J Soc Neurosci 28:4271–4282, 2008; Murdoch et al., J Neurosci 30:9523–9532, 2010; Forni et al., J Neurosci Off J Soc Neurosci 31:6915–6927, 2011b; Freyer et al., Development 138:5403–5414, 2011; Katoh et al., Mol Brain 4:34, 2011). This review analyzes and discusses some recent developmental studies on the OP, placodal derivatives, and olfactory system.

RNAi technology and lentiviral delivery as a powerful tool to suppress Tpr-Met-mediated tumorigenesis

Tpr-Met, the oncogenic counterpart of the Met receptor, has been detected in gastric cancers, as well as in precursor lesions and in the adjacent normal gastric mucosa. This has prompted the suggestion that Tpr-Met may predispose to the development of gastric tumors. Given the sequence specificity of RNA interference, oncogenes activated by point mutation or rearrangements can be targeted while spearing the product of the wild-type allele. In this work, we report specific suppression of Tpr-Met expression and inhibition of Tpr-Met-mediated transformation and tumorigenesis by means of a short interfering RNA (siRNA) directed toward the Tpr-Met junction (anti-TM2). When delivered by a lentiviral vector, anti-TM2 siRNA was effective also in mouse embryonal fibroblasts or epithelial cells expressing high levels of Tpr-Met. Our results suggest that lentiviral-mediated delivery of anti-TM2 siRNA may be developed into a powerful tool to treat Tpr-Met-positive cancers.

Analysis of Mlc-lacZ Met mutants highlights the essential function of Met for migratory precursors of hypaxial muscles and reveals a role for Met in the development of hyoid arch-derived facial muscles

The Pax3 and c‐met genes are necessary for the development of tongue, diaphragm, and limb muscles. These hypaxial muscles derive from precursors that migrate out of the ventrolateral lip of the somites at occipital, cervical, and limb levels. In this work, we re‐examined primary myogenesis in c‐met signaling mutants using a skeletal muscle‐specific lacZ transgene (Mlc3f‐nlacZ‐2E). This strategy allowed us to identify precisely the shoulder, limb, tongue, and dermal muscles that need Met for development and to confirm that the morphological structure of epaxial and body wall muscles was normal, even in the most severe c‐met mutant. Surprisingly, however, X‐gal staining showed that, in this mutant, hyoid arch‐derived facial muscles were either reduced or absent, thus revealing that Met also contributes to the development of muscles in the head. Developmental Dynamics 231:582–591, 2004.

Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity. DOI: http://dx.doi.org/10.7554/eLife.12116.001

A Role for FE65 in Controlling GnRH-1 Neurogenesis

Gonadotropin-releasing hormone-1 (GnRH-1) neurons migrate from the nasal placode to the forebrain where they control gonadal function via the hypothalamic–pituitary–gonadal axis. The birth of GnRH-1-expressing neurons is one of the first neurogenic events in the developing nasal placode. By gene expression screening on single GnRH-1 neurons, amyloid precursor binding protein-1 (FE65) was identified in migratory GnRH-1 neurons. FE65 has been shown to modulate β1-integrin dynamics, actin cytoskeleton, cell motility, and FE65/amyloid precursor protein signaling has been described in neuro/glial cell fate determination as well as in modulating neurogenesis. Analysis of two mouse lines, one deficient for the 97 kDa FE65 isoform and a second deficient for the 97 and 60 kDa forms of FE65, showed overlapping phenotypes. In both lines, no migratory defects of the GnRH-1 neurons were observed, but a 25% increase in GnRH-1 neuronal number during embryonic development was found. Bromodeoxyuridine birth tracing and spatiotemporal tracking of GnRH-1 cell precursors demonstrated that the lack of the N-terminal portion of FE65, which includes part of the functional nuclear translocation/gene transcription domain of FE65 (WW domain), extends the timing of GnRH-1 neurogenesis in the developing nasal placode without affecting proliferation of GnRH-1 neuronal progenitors or cell death. The observed changes in the dynamics of GnRH-1 neurogenesis highlight a unique role for the 97 kDa isoform of FE65 and suggest that GnRH-1 cells, which have a short neurogenic window, originate from multipotent progenitors able to generate distinct cell types as GnRH-1 neurogenesis declines in response to environmental changes.