identified that can promote the formation of rods but the mechanisms by which these signals regulate the transition from progenitor to postmitotic neuron are only partially understood (Altshuler et al. 1993; Levine et al. 2000; Yang 2004).

One of the most robust findings by many groups has been that signal transducer and activator of transcription 3 (STAT3) is a key signaling protein that regulates rod formation. STAT3 is tyrosine phosphorylated in response to a range of factors including epidermal growth factor (EGF), granulocyte colony-stimulating factor (G-CSF), leptin and IL-6 family cytokines such as ciliary neurotrophic factor (CNTF), oncostatin M and leukemia inhibitory factor (LIF). In addition to activating STAT3, the IL-6 family of ligands causes activation of the ERK1/2 members of the MAPK family. We, and others, have shown that IL-6 family ligands are potent inhibitors of rod differentiation (Neophytou et al. 1997; Ezzedine et al. 1997; Zhang et al. 2004). By using adenovirus constructs controlling expressing of dominant negative or constitutively active forms of STAT3, and inhibitors of ERK activation, we showed that the inhibitory action of CNTF was mediated entirely by STAT3 (Zhang et al. 2004). Similar results have been obtained in a number of other studies (Rhee et al. 2004). Because STAT3 is found in proliferating progenitors and then rapidly lost from the postmitotic rods, we suggested that a major action of CNTF and related ligands might be to inhibit exit of daughter cells from the progenitor pool into a differentiation path (Zhang et al. 2005).

There is much less information about the mechanism by which rod formation is induced.

Although a number of factors, as diverse as taurine and IL-4, can increase the number of rods formed in vitro, little is know about the ways in which these effects are achieved (Altshuler et al. 1993; da Silva et al. 2008). We have recently found that protein kinase C (PKC) is a key signal molecule in this process. PKC is a cyclic nucleotide-independent enzyme that phosphorylates serine and threonine residues in many target proteins. There are a number of isozymes of PKC and one group of these shares the property of activation by phorbol esters. Since PKC was first identified, its involvement in many biological processes has been demonstrated, including development, memory, and carcinogenesis. It also triggers several pathways signaling cell proliferation and differentiation.

In this study we show that activation of PKC by phorbol esters leads to an increase in the number of rod photoreceptors in retinal explants. We examined the expression of the phorbol-ester activated isoforms of PKC in the developing retina and found that PKC-beta1, but not PKC alpha or PKC beta2, is present at the right time in the right place to regulate rod formation. One of the ways in which PKC may exert its action is blocking the activation of STAT3.

3.2 Materials and Methods

3.2.1 Reagents

Recombinant mouse LIF was purchased from Chemicon (Temecula, CA), STAT3 (C20) polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz,