Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2

Abstract

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

Fig. 1.

Isl1-deleted optic nerves and retinas have abnormal structures. (A) The optic nerve from Isl1-deleted (MT) eye is significantly thinner than the WT control. (B and C) Transmission electron microscopy on thin sections across the optic nerves. Compared with WT controls (B), the axons in the optic nerve from Isl1-deleted eyes (C) are undermyelinated and disorganized. Arrows point to empty or partially empty myelin enclosures. (D and E) Hematoxylin and eosin staining of retina sections reveal that there are fewer cells in the INL and GCL of Isl1-deleted retinas (E) than in WT controls (D).

Fig. 2.

RGCs are born normally but lost at later stages in Isl1-deleted retinas. Development of RGCs was examined by anti-Pou4f2 or anti-Ina antibody staining (green) with WT (WT) and mutant (MT) retinas at different developmental stages. Red is nuclear staining by propidium iodide (PI). (A–H) There are equivalent numbers of RGCs at E14.5 (A and B) and E17.5 (C–H) in Isl1-deleted and control retinas, and they migrate to the GCL normally. PI staining shows that the GCLs (E and F, indicated by half brackets) in the WT and mutant retina are the same thickness. At E17.5, most RGCs express Pou4f2 at a lower level (D) than in the control (C), but they express Ina at an equivalent level (G and H). (I–L) At P5 and P16, the number of RGCs was dramatically reduced (J and L) compared with the control (I and K). (M and N) At E14.5, an equivalent number of apoptotic cells (green, indicated by white arrowheads) is seen in WT and Isl1-mutant retinas. (O and P) At 17.5, significantly more apoptotic cells are seen in Isl1-deleted retinas compared with that of WT controls; most of the apoptotic cells are located in or near the GCL (P).

Fig. 3.

Expression of a set of RGC genes revealed by immunofluorescence and in situ hybridization. Isl1 regulates a subset of RGC genes distinct from but overlapping with those that depend on Pou4f2. The expression of RGC genes was examined by immunostaining or in situ hybridization on E14.5 WT and mutant retinal sections. Some genes (C–F and O–X) are down-regulated in Isl1-deleted retinas, and others do not change (A, B, and G–N); some of these genes (A, B, E, F, I, J, O, P, S, T, W, and X) depend on Pou4f2 for their expression, whereas others (C, D, K–N, Q, R, U, and V) do not.

Fig. 4.

Comparison of the three gene sets whose expression depends on Math5, Pou4f2, and Isl1. Gene symbols are listed on the left, and the 17 transcription-factor genes are highlighted in red. The three bars in each row represent the changes of each gene in the retinas of the three mutant genotypes (Math5-, Pou4f2-, and Isl1-null), respectively. Significant changes are indicated with red bars, yellow bars mean no significant changes, and gray bars indicate data not available. Note that Isl1 showed no change in Isl1-null retinas, probably because deletion of exon 3 did not affect its mRNA level.

Fig. 5.

A diagram depicting different gene regulation branches for RGC gene expression in the RGC GRN. Isl1 (green) and Pou4f2 (red) define two such parallel branches, which are both downstream of the retinal determination factors Pax6 and Notch (represented by its transcription factor effectors Hes1 and Hes5), and the RGC-specific regulator Math5. The Isl1 and Pou4f2 pathways interact by coregulating a subset of RGC-expressing genes. Isl1 may also regulate Pou4f2 directly (green broken line). Other branch(es) (black line) mediated by other transcription factors such as Tle1 and Myt1 are postulated, and they may also interact with the Isl1 and Pou4f2 branches (represented by broken black lines). Individual RGC genes receive a distinct combination of upstream inputs from these branches to achieve RGC expression. One example each is provided for genes subject to the four possible combinations of upstream inputs.