Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 (-) retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 (-) precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 (kip1) and P27 (kip2) ) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages

A Novel, Sensitive Assay for Behavioral Defects in Parkinson's Disease Model Drosophila

Parkinson's disease is a common neurodegenerative disorder with the pathology of α-synuclein aggregation in Lewy bodies. Currently, there is no available therapy that arrests the progression of the disease. Therefore, the need of animal models to follow α-synuclein aggregation is crucial. Drosophila melanogaster has been researched extensively as a good genetic model for the disease, with a cognitive phenotype of defective climbing ability. The assay for climbing ability has been demonstrated as an effective tool for screening new therapeutic agents for Parkinson's disease. However, due to the assay's many limitations, there is a clear need to develop a better behavioral test. Courtship, a stereotyped, ritualized behavior of Drosophila, involves complex motor and sensory functions in both sexes, which are controlled by large number of neurons; hence, behavior observed during courtship should be sensitive to disease processes in the nervous system. We used a series of traits commonly observed in courtship and an additional behavioral trait—nonsexual encounters—and analyzed them using a data mining tool. We found defective behavior of the Parkinson's model male flies that were tested with virgin females, visible at a much younger age than the climbing defects. We conclude that this is an improved behavioral assay for Parkinson's model flies

Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis.

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types

Pax6 is essential for the generation of late-born retinal neurons and for inhibition of photoreceptor-fate during late stages of retinogenesis.

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types

Increased expression of cell-cycle factors in the <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina.

<p>Expression of Pax6 (A,E,I,M) and of cell-cycle progression and withdrawal factors was monitored using immunofluorescence in control (A–D,I–L) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (E–H,M–P) distal retina at E16 and E17, as indicated. <i>Pax6</i>^- area was delineated by staining for Pax6 protein (E,M) on adjacent sections (dotted line in E–H,M,N). Expression of Ccnd2 (B,F) Ccnd3 (C,G), P57^Kip2 (D,H) and P27^Kip1 (green in J–L,N–P) is upregulated in most of the <i>Pax6</i>^- RPCs of <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina. Ccnd1 and P27^Kip1, which are expressed in different cells in the control (J) are coexpressed in <i>Pax6</i>^- RPCs (N). For the control and Pax6-deficient cells, P27^Kip1-expressing cells are negative for Ki67 (red in K,O) and BrdU (red in L, P). Abbreviations: GCL, ganglion cell layer; NBL, neuroblastic layer. Scale bar in A is 100 µm.</p

Neurogenic Pax6^- RPCs in the <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina display increased cell-cycle exit and sustained expression of cell-cycle factors.

<p>A single pulse of BrdU was administered at E14.5, E15.5, E16.5 and E18.5, 24 h prior to sacrifice as indicated (A, B). Pax6 expression was detected on adjacent sections to identify the recombination area in the <i>Pax6^loxP/loxP</i>;α<i>-Cre</i> OC. (A) The percentage of BrdU ⁺ PCNA^-/BrdU⁺_Total was determined for the Pax6^{<i>loxP/loxP</i>} control (green bars) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (blue bars) distal retina. <i>Pax6</i>^- RPCs show increased cell-cycle exit at all stages tested (19% (SD=0.6%), 21% (SD=2.7%) and 24.1% (SD=2.6%) compared to 16.6% (SD=0.93%), 16.7% (SD=2%) and 15.9% (SD=2%) in control at E14.5, E16.5 and E18.5, respectively; <i>p</i>≤0.05, n≥3). (B) The number of BrdU⁺ and BrdU ⁺ Ki67^- cells was quantified and the ratio BrdU ⁺ Ki67^-/BrdU⁺_Total was used to compare cell-cycle exit rate in control (green bar) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (blue bar) distal retina at E15.5. <i>Pax6</i>^- RPCs show increased cell-cycle exit (37.3% (SD=3.6%) in <i>Pax6</i>^- compared to 14% (SD=1.5%) in control; <i>p</i><0.01, n=6 for both genotypes). Triple immunofluorescence for PCNA, Ki67 and BrdU (red, green and blue, respectively, in C–J) in control (C–F) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;<i>α-Cre</i> (G–J) distal retina showing mitotic PCNA ⁺ Ki67 ⁺ BrdU⁺ in both control and mutant (white circles in D–F,H–J) and abnormal PCNA ⁺ Ki67^-BrdU^- cells detected only in <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (yellow circles in H–J) OC. Scale bars in C,D are 100 and 25 µm, respectively.</p

Scheme depicting <i>Pax6</i> roles during the transition from proliferating retinal progenitor to differentiating retinal neuron.

<p>(A) In normal cycling retinal progenitors, <i>Pax6</i> regulates the balance between proliferation promoting (i.e. <i>Nr2e1</i>, <i>Vsx2</i>, Hedgehog (HH) signaling) and inhibiting factors (i.e. <i>Plagl1</i>). These in turn regulate the expression of genes which induce either progression of (i.e. <i>Ccnd1–3</i>) or withdrawal (<i>P27</i>^<i>Kip1</i>, <i>P57</i>^<i>Kip2</i>) from the cell cycle. It is also required for the expression of bHLH proneural factors (<i>Neurog2</i>, <i>Atoh7</i>, and <i>Ascl1</i>) presumed to inhibit cell-cycle factors as well as promote specific retinal lineages. (B) <i>Pax6</i> loss from RPCs results in aberrant cell-cycle exit as <i>Ccnd1–3</i>, as well as <i>P27</i>^<i>Kip1</i> and <i>P57</i>^<i>Kip2</i>, are elevated and several cell-fate determination factors show reduced (bHLH proneural factors) or increased (<i>Six3</i>, <i>Sox2</i>) expression. The combined outcome of these alterations is delayed differentiation of the <i>Pax6</i>-deficient cells to only one subclass of retinal interneurons.</p

Abrogated expression of factors implicated in regulating RPC proliferation in <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina.

<p>The expression pattern of different factors was monitored on sections from eyes of control (A-D,I,J) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (E-H,K,L) mice. Nr2e1 (E12.5 A,E; E14.5 B,F) and Plagl1 (E12.5 I,K; E14.5 J,L) were detected using ISH while Vsx2 (green; E12.5 C,G; E15.5 D,H) Pax6 (red; E12.5 C,G; E15.5 D,H) were detected by IIF analysis. Pax6 (red in C,D,G,H and not shown) and Crx (inset in E and not shown) expression were used to delineate the different RPC populations in the <i>Pax6^loxP/loxP</i>;α<i>-Cre</i> retina (marked with dotted line in E-H,K,L). Scale bar in F is 100 µm.</p

Hedgehog signaling is disrupted in <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina.

<p>Expression of Gli1 was detected using ISH (B,D,F,H) and Pax6 protein was detected by IIF on adjacent sections (A,C,E,G) for control (A-D) and <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> (E-H) OC. Gli1 expression was abrogated in all <i>Pax6</i>^- RPCs (F,H) compared to controls (B,D) at both E14.5 (B,F) and E18.5 (D,H). Pax6^- area was determined by antibody labeling on adjacent sections and marked by dotted line (E-H). Scale bar in B is 100 µm.</p

Delayed differentiation of amacrine precursors in the <i>Pax6</i>^{<i>loxP/loxP</i>} ;α<i>-Cre</i> retina.

<p>(A) A scheme of the stages of amacrine interneuron differentiation. Amacrine cells evolve from FoxN4-expressing RPCs. In the postmitotic amacrine precursors, FoxN4 is reduced and Ptf1a expression is initiated. The Ptf1a-positive precursors migrate to the prospective INL, lose Ptf1a expression and initiate expression of TFs involved in the differentiation of amacrine subtypes (e.g. Ap2α and bHLHb5). The final differentiation of amacrine cells occurs a few days after birth with accumulation of neurotransmitters and transporters (e.g. GABA, glycine transporter GlyT1). Expression of amacrine specification and differentiation markers in control (B,C,F,G,J,K,N,O) and <i>Pax6^loxP/loxP</i>;<i>α-Cre</i> OC (D,E,H,I,L,M,P,Q). Expression of FoxN4 (B–E, the inset in B and D is Crx on adjacent section) as detected using ISH. Indirect immunofluorescence (IIF) was employed to detect the expression of Ptf1a and Pax6 (green and red, respectively, F–I; adjacent sections to B-E respectively), bHLHb5 and Ap2α (green and red, respectively, J–M) and GABA (green, N–Q) during various stages of development as indicated. Pax6 (red in F–I and not shown) and Crx (inset in B,D and not shown) expression determined by IIF or ISH was used to identify the Pax6-deficient area (yellow line in D,E,H,I,L,M) and to delineate the neurogenic and nonneurogenic RPC populations in the <i>Pax6</i>^loxP/loxP;α<i>-Cre</i> retina (numbered 1 and 2 and separated by a dotted white line in D and H). Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; NBL, neuroblastic layer. Scale bar in C is 100 µm.</p