CD24 Expression Identifies Teratogen-Sensitive Fetal Neural Stem Cell Subpopulations: Evidence from Developmental Ethanol Exposure and Orthotopic Cell Transfer Models

Ethanol is a potent teratogen. Its adverse neural effects are partly mediated by disrupting fetal neurogenesis. The teratogenic process is poorly understood, and vulnerable neurogenic stages have not been identified. Identifying these is a prerequisite for therapeutic interventions to mitigate effects of teratogen exposures.We used flow cytometry and qRT-PCR to screen fetal mouse-derived neurosphere cultures for ethanol-sensitive neural stem cell (NSC) subpopulations, to study NSC renewal and differentiation. The identity of vulnerable NSC populations was validated in vivo, using a maternal ethanol exposure model. Finally, the effect of ethanol exposure on the ability of vulnerable NSC subpopulations to integrate into the fetal neurogenic environment was assessed following ultrasound guided, adoptive transfer.Ethanol decreased NSC mRNAs for c-kit, Musashi-1and GFAP. The CD24(+) NSC population, specifically the CD24(+)CD15(+) double-positive subpopulation, was selectively decreased by ethanol. Maternal ethanol exposure also resulted in decreased fetal forebrain CD24 expression. Ethanol pre-exposed CD24(+) cells exhibited increased proliferation, and deficits in cell-autonomous and cue-directed neuronal differentiation, and following orthotopic transplantation into naïve fetuses, were unable to integrate into neurogenic niches. CD24(depleted) cells retained neurosphere regeneration capacity, but following ethanol exposure, generated increased numbers of CD24(+) cells relative to controls.Neuronal lineage committed CD24(+) cells exhibit specific vulnerability, and ethanol exposure persistently impairs this population's cell-autonomous differentiation capacity. CD24(+) cells may additionally serve as quorum sensors within neurogenic niches; their loss, leading to compensatory NSC activation, perhaps depleting renewal capacity. These data collectively advance a mechanistic hypothesis for teratogenesis leading to microencephaly

Ethanol effects on mRNA transcripts for cytoskeletal proteins.

<p>QRT-PCR analysis of mRNA expression for lineage-specific intermediate filament and microtubule-associated proteins, showing a statistically significant decrease in the expression of GFAP mRNA with ethanol exposure. Asterisks indicate statistically significant differences relative to controls.</p

Time-line.

<p>Schematic of the time-line for the experimental protocol for ultrasound-guided, trans-uterine microinjection of immuno-magnetically isolated CD24⁺ cells.</p

Ethanol effects on stem cell mRNAs.

<p>QRT-PCR analysis of mRNA expression of stem cell markers, expressed as fold-change relative to the mean of control, in control and ethanol treated mouse neurosphere cultures showing a general ethanol-related decrease in stem cell mRNAs and significant decline in c-Kit and Musashi-1 mRNA. Asterisks indicate statistically significant differences relative to controls.</p

In utero orthotopic CD24⁺ cell transfer.

<p>(<b>a1</b>-<b>a3</b>) Sequential series of Ultrasound images showing trajectory of microcapillary injection of CD24⁺ cells into the lateral ventricles of GD13 mice showing pre-injection (<b>a1</b>), insertion (<b>a2</b>) and dilation of lateral ventricles (<b>a3</b>), confirming infusion of cells. Sample photomicrographs of control (<b>b1</b>–<b>2</b>) and ethanol pre-exposed (<b>c1–3</b>) CD24⁺ cells microinjected into a naïve GD13 fetal brain. Control immuno-magnetically isolated CD24⁺ cells integrate into the ventricular zone (VZ) after trans-uterine ultrasound guided microinjection, whereas ethanol pre-treated cells localize preferentially to the ventricles (<b>c1</b>–<b>2,</b> ventricles are delineated by white dashed lines) or adhere to the apical region of the VZ (<b>d</b>). (<b>e</b>,<b>f</b>) Quantitative analysis shows that total number of surviving (<b>e</b>) and integrated (<b>f</b>) CD24⁺ cells is significantly decreased in the ethanol pre-exposure condition compared to controls. Asterisks indicate statistically significant differences relative to controls. Scale bar, <b>b-c</b>, 50 µm.</p

List of primers for qRT-PCR analysis

<p>List of primers for qRT-PCR analysis</p

Characterization of immuno-magnetically selected CD24⁺ and CD24^depleted populations.

<p>(a) Flow cytometric frequency histogram of CD24⁺ (top panel) and CD24^depleted (middle panel) cells immuno-magnetically selected from control and ethanol-exposed cultures. ‘X’-axis shows CD24 fluorescence intensity and ‘Y’-axis shows cell number. Bottom panel shows background labeling with isotype-specific control antibody. (<b>b–e</b>) Photomicrographs of control CD24⁺ and CD24^depleted cells maintained for seven days in mitogenic medium (<b>b</b>,<b>c</b>) show that CD24⁺ cells do not form neurospheres in culture (<b>b</b>) but do undergo mitosis (<b>c</b>) and form small adherent cell colonies (<b>d</b>), whereas CD24^depleted cells form large neurospheres (<b>e</b>). Scale bar, <b>b</b>,<b>e</b>, 100 µm; <b>c</b>,<b>d</b>, 50 µm.</p

Ethanol prevents CD24⁺ cell differentiation.

<p>Photomicrographs of Map2a/b immuno-labeled control CD24^depleted (<b>a</b>) compared to control (<b>b</b>,<b>c</b>) and ethanol-pretreated (<b>d</b>,<b>e</b>) CD24⁺ immuno-magnetically isolated cells following mitogen-withdrawal induced differentiation. Cells are counterstained with DAPI (blue fluorescence) to visualize nuclei. Control, CD24^depleted cells do not undergo morphological transformation and exhibit near undetectable levels of Map2a/b immunofluorescence, showing that they are not committed to the neuronal lineage following mitogen withdrawal. Compared to control CD24⁺ cells, ethanol pre-treated CD24⁺ cells exhibited decreased neurite outgrowth and little Map2a/b immunofluorescence suggesting that they were resistant to mitogen withdrawal-induced, i.e., cell-autonomous differentiation. Scale bar, <b>a–e</b>, 25 µm.</p