Park Ko Uoon, Randazzo Grace, Jones Kenneth L, Brzezinski Joseph A
Department of Ophthalmology, University of Colorado Denver, Aurora, Colorado, United States.
Department of Pediatrics, Section Hematology/Oncology, University of Colorado Denver, Aurora, Colorado, United States.
Invest Ophthalmol Vis Sci. 2017 Feb 1;58(2):1137-1150. doi: 10.1167/iovs.16-19767.
How retinal bipolar cell interneurons are specified and assigned to specialized subtypes is only partially understood. In part, this is due to a lack of early pan- and subtype-specific bipolar cell markers. To discover these factors, we identified genes that were upregulated in Blimp1 (Prdm1) mutant retinas, which exhibit precocious bipolar cell development.
Postnatal day (P)2 retinas from Blimp1 conditional knock-out (CKO) mice and controls were processed for RNA sequencing. Genes that increased at least 45% and were statistically different between conditions were considered candidate bipolar-specific factors. Candidates were further evaluated by RT-PCR, in situ hybridization, and immunohistochemistry. Knock-in Tmem215-LacZ mice were used to better trace retinal expression.
A comparison between Blimp1 CKO and control RNA-seq datasets revealed approximately 40 significantly upregulated genes. We characterized the expression of three genes that have no known function in the retina, Gsg1 (germ cell associated gene), Trnp1 (TMF-regulated nuclear protein), and Tmem215 (a predicted transmembrane protein). Germ cell associated gene appeared restricted to a small subset of cone bipolars while Trnp1 was seen in all ON type bipolar cells. Using Tmem215-LacZ heterozygous knock-in mice, we observed that β-galactosidase expression started early in bipolar cell development. In adults, Tmem215 was expressed by a subset of ON and OFF cone bipolar cells.
We have identified Gsg1, Tmem215, and Trnp1 as novel bipolar subtype-specific genes. The spatial and temporal pattern of their expression is consistent with a role in controlling bipolar subtype fate choice, differentiation, or physiology.
视网膜双极细胞中间神经元如何被确定并分配到特定的亚型，目前仅得到部分理解。部分原因在于缺乏早期的泛双极细胞和亚型特异性标记物。为了发现这些因素，我们鉴定了在Blimp1（Prdm1）突变视网膜中上调的基因，该突变视网膜表现出双极细胞发育早熟。
对出生后第2天（P2）的Blimp1条件性敲除（CKO）小鼠和对照小鼠的视网膜进行RNA测序。在不同条件下增加至少45%且具有统计学差异的基因被视为候选双极细胞特异性因子。通过逆转录聚合酶链反应（RT-PCR）、原位杂交和免疫组织化学对候选基因进行进一步评估。使用敲入Tmem215-LacZ小鼠来更好地追踪视网膜表达情况。
Blimp1 CKO和对照RNA测序数据集之间的比较揭示了约40个显著上调的基因。我们对视网膜中三个功能未知的基因进行了表征，即生殖细胞相关基因（Gsg1）、TMF调节核蛋白（Trnp1）和预测的跨膜蛋白（Tmem215）。生殖细胞相关基因似乎局限于一小部分视锥双极细胞，而Trnp1在所有ON型双极细胞中都有表达。使用Tmem215-LacZ杂合敲入小鼠，我们观察到β-半乳糖苷酶表达在双极细胞发育早期开始。在成年小鼠中，Tmem215由一部分ON和OFF视锥双极细胞表达。
我们已将Gsg1、Tmem215和Trnp1鉴定为新型双极细胞亚型特异性基因。它们表达的时空模式与在控制双极细胞亚型命运选择、分化或生理功能方面的作用一致。
