Analysis of hGFAP:CKO mice using the Aldh1l1-EGFP transgene shows presence of dysregulated astrocytes with increase EGFP signals.

<p>(a and b) P16 hGFAP:CKO forebrains have decreased sizes. (c,d, and e) P16 cortex and (f, g, and h) spinal cord sections indicate the presence of Aldh1l1-EGFP positive cells with higher EGFP signal in the hGFAP:CKO CNS. (i) The increase in EGFP signals can be visualized using FACS (blue represents P16 wildtype and dotted black represents P16 hGFAP:CKO Aldh1l1-EGFP positive cells). X axis in i represents EGFP levels in log2 scale. Y axis represents cell numbers. Scale bars in a and b represent 5 mm. Scale bars in c, d, and e represent 50 μm. Scale bars in f, g, and h represent 100 μm. FACS data for panel i is representative of 3 experiments. All histological experiments are representative data based on 3 independent observations.</p

EGFP positive cells are disorganized and have increased EGFP signals in mGFAP:CKO cerebellums.

<p>(a) In symptomatic 2 month old mGFAP:CKO mice, massive cell loss was observed in mutant cerebellum (arrows indicate missing cells in cerebellar lobes). (b-d) EGFP positive cells had up-regulated signals and appeared in the molecular layers in the mutant cerebellums. (e) qRT-PCR also indicated up-regulated levels of <i>Aldh1l1</i> transcripts (p-value< 0.01, n = 3). (e) <i>Gfap</i> showed an upward trend suggesting astrogliosis (p-value = 0.05, n = 3). (e) <i>Aqp4</i> levels were similar between wildtype and mutant animals (p-value = 0.14, n = 3). (e) <i>Glast</i> level was decreased in mutant cerebellums (p-value < 0.01, n = 3). (f-h) In P16 mGFAP:CKO, defects with Aldh1l1-EGFP could already be observed in the cerebellum. Aldh1l1-EGFP cells were present in mutant molecular layer. (i-k) While ki67 positive cells were limited in wildtype cerebellum, we saw a noticeable increase in mutant cerebellum (ki67 cells are red). (l-m) These ki67 positive cells co-localized predominately with EGFP cells. <i>Aldh1l1</i> and <i>Gfap</i> transcripts were up-regulated while <i>Glast</i> had a small decrease expression level in mutant cerebellum. (o) No change was detected in <i>Aqp4</i> (<i>Aldh1l1</i> p-value< 0.01, <i>Aqp4</i> p-value = 0.16, <i>Gfap</i> p-value< 0.05, and <i>Glast</i> p-value< 0.05, n = 3). Scale bars in a-d and f-n represent 50 μm. Gray bars represent wildtype mice and yellow bars represent mGFAP:CKO mice in e and o. Asterisks in e and o indicate significant level (** represents p< 0.01 and * represents p< 0.05). Y axes in e and o represent the levels for gene of interest normalized to <i>Gapdh</i>. The average expression level of wildtype samples were normalized to 1. Unpaired t-test was used to determine p-values.</p

Understanding the Role of Dicer in Astrocyte Development

<div><p>The <i>Dicer1</i> allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the <i>in vivo</i> phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional <i>Dicer1</i> ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models.</p></div

Genes with >2-fold up-regulation in hGFAPCKO Aldh1l1-EGFP that are also up- regulated in primary cultures.

<p>Genes with >2-fold up-regulation in hGFAPCKO Aldh1l1-EGFP that are also up- regulated in primary cultures.</p

Pathway enriched in wildtype FACS cells compare to hGFAP:CKO FACS cells.

<p>Pathway enriched in wildtype FACS cells compare to hGFAP:CKO FACS cells.</p

qRT-PCR indicates that FACS isolated hGFAP:CKO Aldh1l1-EGFP cells exhibit immature molecular phenotype.

<p>qRT-PCR indicated mutant cells have significantly down-regulated expression of <i>Aqp4</i> and <i>Glast (p-value< 0</i>.<i>05)</i>. The expression of <i>Gfap</i> did not reach significant down-regulation (p-value of <i>Gfap</i> is 0.09). <i>Fgfr3</i> levels appeared similar and <i>Nes</i> levels were up-regulated (p-value < 0.05). As expected, an amplicon that detected exon 23 of <i>Dicer1</i> had dramatically reduced levels in the mutant cells (p-values < 0.01). Unpaired t-test was used to determine p-values. Gray bars represent wildtype and yellow bars represent hGFAP:CKO. Asterisks indicate significant level (** represents p< 0.01 and * represents p< 0.05). Y axis represents expression levels for the gene of interest normalized to <i>Gapdh</i>. The average expression level of wildtype samples were normalized to 1. N = 3</p