Can zebrafish be used as a model to study the neurodevelopmental causes of autism?

The zebrafish has proven to be an excellent model for analyzing issues of vertebrate development. In this review we ask whether the zebrafish is a viable model for analyzing the neurodevelopmental causes of autism. In developing an answer to this question three topics are considered. First, the general attributes of zebrafish as a model are discussed, including low cost maintenance, rapid life cycle and the multitude of techniques available. These techniques include large-scale genetic screens, targeted loss and gain of function methods, and embryological assays. Second, we consider the conservation of zebrafish and mammalian brain development, structure and function. Third, we discuss the impressive use of zebrafish as a model for human disease, and suggest several strategies by which zebrafish could be used to dissect the genetic basis for autism. We conclude that the zebrafish system could be used to make important contributions to understanding autistic disorders.

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism.

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Retinal stem cells in the adult mammalian eye.

The mature mammalian retina is thought to lack regenerative capacity. Here, we report the identification of a stem cell in the adult mouse eye, which represents a possible substrate for retinal regeneration. Single pigmented ciliary margin cells clonally proliferate in vitro to form sphere colonies of cells that can differentiate into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia. Adult retinal stem cells are localized to the pigmented ciliary margin and not to the central and peripheral retinal pigmented epithelium, indicating that these cells may be homologous to those found in the eye germinal zone of other nonmammalian vertebrates.

Separate proliferation kinetics of fibroblast growth factor-responsive and epidermal growth factor-responsive neural stem cells within the embryonic forebrain germinal zone.

The embryonic forebrain germinal zone contains two separate and additive populations of epidermal growth factor (EGF)- and fibroblast growth factor (FGF)-responsive stem cells that both exhibit self-renewal and multipotentiality. Although cumulative S phase labeling studies have investigated the proliferation kinetics of the overall population of precursor cells within the forebrain germinal zone through brain development, little is known about when and how (symmetrically or asymmetrically) the small subpopulations of stem cells are proliferating in vivo. This has been determined by injecting timed-pregnant mice with high doses of tritiated thymidine ((3)H-thy) to kill any stem cells proliferating within the striatal germinal zone in vivo and then by assaying for neurosphere formation in vitro. Injections of 0.8 mCi of (3)H-thy given every 2 hr for 12 hr to timed-pregnant mice at E11, E14, and E17 resulted in significant depletions in the number of neurospheres generated by FGF-responsive stem cells at E11 and by EGF-responsive and FGF-responsive stem cells at E14 and E17. With increasing embryonic age, the depletions observed in the number of neurospheres generated in vitro in response to FGF2 after exposure to (3)H-thy in vivo decreased, suggesting there is an increase in the length of the cell cycle of FGF-responsive neural stem cells through embryonic development. The results suggest that the FGF-responsive stem cell population expands between E11 and E14 by dividing symmetrically, but switches to primarily asymmetric division between E14 and E17. The EGF-responsive stem cells arise after E11, and their population expands through symmetric divisions and through asymmetric divisions of FGF-responsive stem cells.

Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.

The adult derivatives of the embryonic forebrain germinal zones consist of two morphologically distinct cell layers surrounding the lateral ventricles: the ependyma and the subependyma. Cell cycle analyses have revealed that at least two proliferating populations exist in this region, one that is constitutively proliferating and one that is relatively quiescent and thought to include the endogenous adult neural stem cells. Earlier studies demonstrated that specific dissection of the region surrounding the lateral ventricles was necessary for the in vitro isolation of multipotent, self-renewing neural stem cells. However, in these studies, the ependymal layer was not physically separated from the subependymal layer to identify the specific adult laminar localization of the neural stem cells around the lateral ventricles. To determine which cellular compartment in the adult forebrain contained the neural stem cells, we isolated and cultured the ependyma separately from the subependyma and tested for the presence of neural stem cells using the in vitro neurosphere assay. We demonstrate that the ependymal cells can proliferate in vitro to form sphere-like structures. However, the ependymal cells generating spheres do not have the ability to self-renew (proliferate to form secondary spheres after dissociation) nor to produce neurons, but rather only seem to generate glial fibrillary acidic protein-positive ependymal cells when plated under differentiation conditions in culture. On the other hand, a subpopulation of subependymal cells do possess the self-renewing and multipotential characteristics of neural stem cells. Therefore, the adult forebrain neural stem cell resides within the subependymal compartment.

Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon.

Multipotent, self-renewing neural stem cells reside in the embryonic mouse telencephalic germinal zone. Using an in vitro neurosphere assay for neural stem cell proliferation, we demonstrate that FGF-responsive neural stem cells are present as early as E8.5 in the anterior neural plate, but EGF-responsive neural stem cells emerge later in development in a temporally and spatially specific manner. By separately blocking EGF and FGF2 signaling, we also show that EGF alone and FGF2 alone can independently elicit neural stem cell proliferation and at relatively high cell densities separate cell nonautonomous effects can substantially enhance the mitogen-induced proliferation. At lower cell densities, neural stem cell proliferation is additive in the presence of EGF and FGF2 combined, revealing two different stem cell populations. However, both FGF-responsive and EGF-responsive neural stem cells retain their self-renewal and multilineage potential, regardless of growth factor conditions. These results support a model in which separate, lineage-related EGF- and FGF-responsive neural stem cells are present in the embryonic telencephalic germinal zone.

Transforming growth factor-alpha null and senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma.

The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-alpha (TGFalpha) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23- to 25-months-old compared with 2- to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGFalpha does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGFalpha and the effects of senescence may regulate the proliferation of progenitor cells in the adult subependyma, but that the number of neural stem cells is maintained throughout life.

In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.

The lateral ventricle subependyma in the adult mammalian forebrain contains both neural stem and progenitor cells. This study describes the in situ modulation of these subependymal neural precursor populations after intraventricular administration of exogenous growth factors. In vivo infusion of epidermal growth factor (EGF) into adult mouse forebrain for 6 consecutive days resulted in a dramatic increase in the proliferation and total number of subependymal cells and induced their migration away from the lateral ventricle walls into adjacent parenchyma. Immediately after EGF infusion, immunohistochemical characterization of the EGF-expanded cell population demonstrated that >95% of these cells were EGF receptor- and nestin-positive, whereas only 0.9% and 0.2% labeled for astrocytic and neuronal markers, respectively. Seven weeks after EGF withdrawal, 25% of the cells induced to proliferate after 6d of EGF were still detectable; 28% of these cells had differentiated into new astrocytes and 3% into new neurons in the cortex, striatum, and septum. Newly generated oligodendrocytes were also observed. These in vivo results (1) confirm the existence of EGF-responsive subependymal neural precursor cells in the adult mouse forebrain and (2) suggest that EGF acts directly as a proliferation, survival, and migration factor for subependymal precursor cells to expand these populations and promote the movement of these cells into normal brain parenchyma. Thus, in situ modulation of endogenous forebrain precursor cells represents a novel model for studying neural development in the adult mammalian brain and may provide insights that will achieve adult replacement of neurons and glia lost to disease or trauma.