Sim1 Is a Novel Regulator in the Differentiation of Mouse Dorsal Raphe Serotonergic Neurons

BACKGROUND: Mesencephalic dopaminergic neurons (mDA) and serotonergic (5-HT) neurons are clinically important ventral neuronal populations. Degeneration of mDA is associated with Parkinson's disease; defects in the serotonergic system are related to depression, obsessive-compulsive disorder, and schizophrenia. Although these neuronal subpopulations reveal positional and developmental relationships, the developmental cascades that govern specification and differentiation of mDA or 5-HT neurons reveal missing determinants and are not yet understood. METHODOLOGY: We investigated the impact of the transcription factor Sim1 in the differentiation of mDA and rostral 5-HT neurons in vivo using Sim1-/- mouse embryos and newborn pups, and in vitro by gain- and loss-of-function approaches. PRINCIPAL FINDINGS: We show a selective significant reduction in the number of dorsal raphe nucleus (DRN) 5-HT neurons in Sim1-/- newborn mice. In contrast, 5-HT neurons of other raphe nuclei as well as dopaminergic neurons were not affected. Analysis of the underlying molecular mechanism revealed that tryptophan hydroxylase 2 (Tph2) and the transcription factor Pet1 are regulated by Sim1. Moreover, the transcription factor Lhx8 and the modulator of 5-HT(1A)-mediated neurotransmitter release, Rgs4, exhibit significant higher expression in ventral hindbrain, compared to midbrain and are target genes of Sim1. CONCLUSIONS: The results demonstrate for the first time a selective transcription factor dependence of the 5-HT cell groups, and introduce Sim1 as a regulator of DRN specification acting upstream of Pet1 and Tph2. Moreover, Sim1 may act to modulate serotonin release via regulating RGS4. Our study underscores that subpopulations of a common neurotransmitter phenotype use distinct combinations of transcription factors to control the expression of shared properties

Evaluation of intensity modulated radiation therapy dose painting for localized prostate cancer using 68 Ga-HBED-CC PSMA-PET/CT: A planning study based on histopathology reference

AbstractPurposeTo demonstrate the feasibility and to evaluate the tumour control probability (TCP) and normal tissue complication probability (NTCP) of IMRT dose painting using 68Ga-HBED-CC PSMA PET/CT for target delineation in prostate cancer (PCa).Methods and materials10 patients had PSMA PET/CT scans prior to prostatectomy. GTV-PET was generated on the basis of an intraprostatic SUVmax of 30%. Two IMRT plans were generated for each patient: Plan77 which consisted of whole-prostate IMRT to 77Gy, and Plan95 which consisted of whole-prostate IMRT to 77Gy and a simultaneous integrated boost to the GTV-PET up to 95Gy (35 fractions). The feasibility of these plans was judged by their ability to adhere to the FLAME trial protocol. TCP-histo/-PET were calculated on co-registered histology (GTV-histo) and GTV-PET, respectively. NTCPs for rectum and bladder were calculated.ResultsAll plans reached prescription doses whilst adhering to dose constraints. In Plan77 and Plan95 mean doses in GTV-histo were 75.8±0.3Gy and 96.9±1Gy, respectively. Average TCP-histo values for Plan77 and Plan95 were 70% (range: 15–97%), and 96% (range: 78–100%, p77 and Plan95 were 55% (range: 27–82%), and 100% (range: 99–100%, p95 (p=0.25). There were no significant differences in rectal (p=0.563) and bladder (p=0.3) NTCPs.ConclusionsIMRT dose painting using PSMA PET/CT was technically feasible and resulted in significantly higher TCPs without higher NTCPs

T‐cell prolymphocytic leukemia is associated with deregulation of oncogenic microRNAs on transcriptional and epigenetic level

Deregulation of micro(mi)-RNAs is a common mechanism in tumorigenesis. We investigated the expression of 2083 miRNAs in T-cell prolymphocytic leukemia (T-PLL). Compared to physiologic CD4+ and CD8+ T-cell subsets, 111 miRNAs were differentially expressed in T-PLL. Of these, 33 belonged to miRNA gene clusters linked to cancer. Genomic variants affecting miRNAs were infrequent with the notable exception of copy number aberrations. Remarkably, we found strong upregulation of the miR-200c/-141 cluster in T-PLL to be associated with DNA hypomethylation and active promoter marks. Our findings suggest that copy number aberrations and epigenetic changes could contribute to miRNA deregulation in T-PLL

Differential regulation of serotonergic lineage determinants by <i>Sim1</i>.

<p>Quantitative real-time PCR analysis of <i>Gata2</i> (A), <i>Mash1</i> (B), <i>Pet1</i> (C) and <i>Tph2</i> (D) mRNA levels in pcDNA3::Sim1-transfected MN9D cells and in pcDNA3-transfected cells. Expression of individual gene is shown as 2^−ΔΔCt±s. <i>Gata2</i> and <i>Mash1</i> expression remained unchanged in pcDNA3::Sim1-transfected MN9D cells, compared to pcDNA3-transfected cells 24 hours, 48 hours, and 72 hours after transfection. In contrast, <i>Pet1</i> and <i>Tph2</i> expression was significantly up-regulated in pcDNA3::Sim1-transfected MN9D cells, compared to the controls 48 hours and 72 hours after transfection (*<i>p</i><0.05; **<i>p</i><0.01; and ***p<0.0001 using the Student's <i>t</i>-test, n = 3).</p

Identification of candidate genes involved in the development of serotonergic neurons.

<p>A: Quantitative real-time PCR analysis showed that expression of the transcription factors <i>Brn3.2</i> and <i>Lhx8</i>, and of a regulator of G protein signalling and modulator of 5-HT_1A-mdiated neurotransmitter release, <i>RGS4,</i> are significantly upregulated in mouse ventral hindbrain tissue compared to ventral midbrain primary tissue at embryonic day 11.5. B-C: Regulation of expression of candidate genes by Sim1, assessed by quantitative real-time PCR. <i>Rgs4</i> (B) and <i>Brn3.2</i> (C) mRNA levels in pcDNA3::Sim1-transfected MN9D cells and in pcDNA3-transfected cells were analyzed. Expression of individual gene is shown as 2^−ΔΔCt±s. <i>Rgs4</i> and <i>Brn3.2</i> expression was significantly up-regulated 24 hours and 48 hours, respectively, after transfection of MN9D cells with pcDNA3::Sim1 expression vector, compared with the controls (**<i>P</i><0.01, using the Student's <i>t</i>-test, n = 3). D: Immunoblotting for RGS4 and Brn3.2 protein abundance in homogenates of MN9D cells transfected either with pcDNA3::Sim1 or pcDNA3 expression vectors (ctl) 48 hours after transfection. The immunoblots were probed either with monoclonal antibody against GAPDH or with a goat polyclonal antibody against RGS4 or Brn3.2. RGS4 protein expression was comparable between controls and pcDNA3::Sim1-transfected MN9D cells. In contrast, Brn3.2 protein abundance was significantly upregulated in pcDNA3::Sim1-transfected MN9D cells, compared to the pcDNA3-transfected cells. (**<i>p</i><0.01 after densitometric analysis of the signal ratio Brn3.2: GAPDH and Student's <i>t</i>-test; n = 3). The blots are representative for three different experiments. 30 µg protein was loaded per lane. E, G: In situ hybridization for expression of candidate genes in mouse at E14.5 using antisense probes. <i>Brn3.2</i> expression was present in hindbrain, but not in the area of rostral serotonergic neurons (E), whereas <i>RGS4</i> expression was detectable in rostral serotonergic neurons (G). F: immunofluorescence for 5-HT at E14.5. H, J: In situ hybridization for expression of candidate genes in mouse at E18.5 using antisense probes. <i>Brn3.2</i> expression was present in hindbrain, but not in the area of rostral serotonergic neurons (H), whereas <i>RGS4</i> expression was detectable in rostral serotonergic neurons (J). I: immunofluorescence for 5-HT at E18.5. K, M: In situ hybridization at E18.5 for expression of <i>Brn3.2</i> (K) and <i>RGS4</i> (M) using sense probes revealed no detectable staining. L: schematic presentation of a sagittal section of mouse brain at E18.5 with a line drawn to indicate the approximate level of brain sections used for H-M (r: rostral, c: caudal). Aq: aqueduct.</p

<i>Sim1</i> loss-of-function <i>in vitro</i> and <i>in vivo</i>.

<p>A: Quantitative real-time PCR analysis for candidate genes in MN9D cells transfected either with control siRNA (ctl) or with specific <i>Sim1</i> siRNA. Gene expression is shown as 2^−ΔΔCt±s. <i>Pet1, Gata2, Brn3.2,</i> and <i>Tph2</i>, but not <i>RGS4</i> expression was significantly down-regulated in <i>Sim1</i> siRNA-transfected cells, compared to the controls, 24 hours after transfection (*<i>p</i><0.05 and **<i>p</i><0.01, using the Student's <i>t</i>-test, n = 3). B-M: In situ hybridization for <i>Pet1</i> (B-E), <i>RGS4</i> (F-I) and <i>Brn3.2</i> (J-M) in wild type (<i>wt</i>) and <i>Sim1-/-</i> mutants at P0. <i>Pet1</i> expression was observed in the area of rostral 5-HT neurons (B) including the area of dorsal raphe in <i>wt</i> (C: magnification of the inset in B), but was considerably decreased in <i>Sim1-/-</i> (D). E: magnification of the inset illustrating the area of the dorsal raphe. <i>RGS4</i> (F, and magnification of the inset in G) was also expressed in the dorsal raphe in <i>wt</i>, and was considerably decreased in <i>Sim1</i> mutants (H and magnification of the inset in I). In contrast, although <i>Brn3.2</i> expression in <i>wt</i> was present (J, asterisks) the area of rostral 5-HT neurons was devoid of <i>Brn3.2</i> expression (J, arrow and magnification of the inset in K). In <i>Sim1-/-</i> mutants <i>Brn3.2</i> expression was absent (L, and magnification of the inset in M). N: immunoblot analysis for Pet1, RGS4, and Brn3.2 in hindbrain of <i>wt</i> and <i>Sim1-/-</i> mutants. Pet1, RGS4, and Brn3.2 protein abundance was significantly downregulated in <i>Sim1-/-</i> hindbrain, compared to <i>wt.</i> (*<i>p</i><0.05 after densitometric analysis of the signal ratio Pet1:GAPDH, RGS4:GAPDH or Brn3.2:GAPDH and Student's <i>t</i>-test; n = 4). The blots are representative for four different experiments. 30 µg protein was loaded per lane.</p

Sim1 mRNA expression by RT-PCR and cellular localization of Sim1 in mouse tissues by immunohistochemistry.

<p>A: <i>Sim1</i> transcript was detected in mouse E12 ventral hindbrain, ventral midbrain, and isthmus region using <i>sim1</i> primers corresponding to nucleotides (nt) 736-756 and nt:1052-1032 of the mouse Sim1 gene (Genbank accession number: NM011376). B: double immunolabeling on mouse fixed paraffin sections for TH (blue) and Sim1 (brown) showed Sim1 localization in the cell nucleus and cytoplasmic staining pattern for TH in midbrain substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Inset at higher magnification shown in C: co-localization of TH and Sim1 is apparent. Arrows point to cells that are immunopositive for both TH and Sim1. Asterisks indicate Sim1 positive, but TH negative cells. D, E: double immunofluorescence for 5-HT (green) and Sim1 (red) in mouse hindbrain revealed co-localization of the proteins.</p

Primer sequences used for RT-PCR gene expression analysis.

<p>(nt: nucleotide).</p