PURPOSE. The early transcriptional events that occur in newly generated cone photoreceptors are not well described. Knowledge of these events is critical to provide benchmarks for in vitro-derived cone photoreceptors and to understand the process of cone and rod photoreceptor diversification. We sought to identify genes with differential gene expression in embryonic mouse cone photoreceptors. METHODS. The specificity of expression of the LHX4 transcription factor in developing cone photoreceptors was examined using immunofluorescence visualization in both mouse and chicken retinas. A LHX4 transgenic reporter line with high specificity for developing mouse cone photoreceptors was identified and used to purify early-stage cone photoreceptors for profiling by single-cell RNA sequencing. Comparisons were made to previous datasets targeting photoreceptors. RESULTS. The LHX4 transcription factor and a transgenic reporter were determined to be highly specific to early developing cone photoreceptors in the mouse. Single-cell transcriptional profiling identified new genes with enriched expression in cone photoreceptors relative to concurrent cell populations. Comparison to previous profiling datasets allowed for further characterization of these genes across developmental time, species, photoreceptor type, and gene regulatory network. CONCLUSIONS. The LHX4 gene is highly enriched in developing cone photoreceptors as are several new genes identified through transcriptional profiling, some of which are expressed in subclusters of cones. Many of these cone-enriched genes do not show obvious de-repression in profiling of retinas mutant for the rod-specific transcription factor NRL, highlighting differences between endogenous cones and those induced in NRL mutants.
26Cone photoreceptors are the critical first cells that mediate high acuity vision. Despite 27 their importance and their potential use in cell-based therapies for retinal diseases, 28 there is a lack of knowledge about the early developmental stages of these cells. Here 29 we characterize the expression of the homeobox transcription factor Lhx4 as an early 30 and enriched cone photoreceptor expressed gene in both chicken and mouse. A Lhx4 31 GFP reporter mouse was found to recapitulate this early cone photoreceptor expression 32 and was used to purify and profile embryonic mouse cone photoreceptors by single cell 33 RNA sequencing. This enrichment in cone photoreceptors allowed for the robust 34 identification of genes associated with the early cone transcriptome and also identified 35 subpopulations of these cells. A comparison to previously reported datasets allowed the 36 classification of genes according to developmental timing, cell type specificity, and 37 whether they were regulated by the rod transcription factor Nrl. This analysis has 38 extended the set of known early cone enriched genes and identified those that are 39 regulated independently of Nrl. This report furthers our knowledge of the transcriptional 40 events that occur in early cone photoreceptors. 41 42 43 44 86 are not completely transformed into cones.87 88 89 RESULTS 90 LHX4 is present in early cone photoreceptors in the chicken retina 91 Recently, we established the transcriptional profile of retinal progenitor cells 92 (RPCs), defined by the activity of the ThrbCRM1 element, that are biased towards the 93 cone and horizontal cell (HC) fate in the early chick retina 3 . Using this dataset, we 94 screened for potential cone-enriched transcripts that could serve as markers for early 95 cones. After establishing a criterion for >1.5-fold change score between cone/HC RPCs 96 and other concurrent populations (enriched in "Other early retinal progenitors") we 97 selected for transcription factors (TFs) enriched in the cone/HC RPCs (Fig 1 A). We 98 identified the LIM homeobox 4 (LHX4) gene as highly enriched, along with known TFs in 99 this population such as THRB, ONECUT1, and OTX2. This transcript has significant fold 100 change (b = 3.3) and a low number of reads in the non-ThrbCRM1 active population, 101suggesting high specificity towards the cone/HC RPC population at this time (Fig 1 B). 102 A previous report has examined the presence of LIM-domain factors in early 103 chick photoreceptor development 15 . This study suggested that LHX3 was abundantly 104 present in the apical portion of the retina and localized to photoreceptors once the ONL 105 is clearly distinguished. As the RNA-Seq data indicated that LHX4 expression is 106 prominent in ThrbCRM1 reporter-positive cells at early stages while LHX3 transcript 107presence is marginal in all targeted cells (Supp. Fig. 1 A), we suspected that this 108 previous study could have detected LHX4 instead of LHX3 at earlier timepoints. To test 109 this, we electroporated a mouse LHX4 misex...
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