SP8 and SP9 coordinately promote D2-type medium spiny neuron production by activating Six3 expression.

Dopamine receptor DRD1-expressing medium spiny neurons (D1 MSNs) and dopamine receptor DRD2-expressing medium spiny neurons (D2 MSNs) are the principal projection neurons in the striatum, which is divided into dorsal striatum (caudate nucleus and putamen) and ventral striatum (nucleus accumbens and olfactory tubercle). Progenitors of these neurons arise in the lateral ganglionic eminence (LGE). Using conditional deletion, we show that mice lacking the transcription factor genes Sp8 and Sp9 lose virtually all D2 MSNs as a result of reduced neurogenesis in the LGE, whereas D1 MSNs are largely unaffected. SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants. Finally, ChIP-Seq reveals that SP9 directly binds to the promoter and a putative enhancer of Six3 Thus, this study defines components of a transcription pathway in a regionally restricted LGE progenitor domain that selectively drives the generation of D2 MSNs.

Expression and Functional Analyses of Ectodermal Transcription Factors FoxJ-r, SoxF, and SP8/9 in Early Embryos of the Ascidian Halocynthia roretzi.

The spatiotemporal expression of zygotic genes is regulated by transcription factors, which mediate cell fate decision and morphogenesis. Investigation of the expression patterns and their transcriptional regulatory relationships is crucial to understand embryonic development. Staged RNA-seq of the ascidian Halocynthia roretzi has previously shown that nine genes encoding transcription factors are transiently expressed at the blastula stage, which is the stage at which cell fates are specified and differentiation starts. Six of these transcription factors have already been found to play important roles during early development. However, the functions of the other transcription factors (FoxJ-r, SoxF, and SP8/9) remain unknown. The study of the spatial and temporal expression patterns showed that all three genes were expressed in the animal hemisphere as early as the 16-cell stage. This is likely due to transcription factor genes that are expressed in the vegetal hemisphere, which have been extensively and comprehensively analyzed in previous studies of ascidians. Functional analyses using FoxJ-r morphants showed that they resulted in the disruption of laterality and the absence of epidermal mono-cilia, suggesting FoxJ-r functions in cilia formation and, consequently, in the generation of left-right asymmetry, as observed in vertebrates. SoxF knockdown resulted in incomplete epiboly by the ectoderm during gastrulation, while SP8/9 knockdown showed no phenotype until the tailbud stage in the present study, although it was expressed during blastula stages. Our results indicate that transcription factor genes expressed at the cleavage stages play roles in diverse functions, and are not limited to cell fate specification.

Integrating evolutionarily novel horns within the deeply conserved insect head.

BACKGROUND: How novel traits integrate within ancient trait complexes without compromising ancestral functions is a foundational challenge in evo-devo. The insect head represents an ancient body region patterned by a deeply conserved developmental genetic network, yet at the same time constitutes a hot spot for morphological innovation. However, the mechanisms that facilitate the repeated emergence, integration, and diversification of morphological novelties within this body region are virtually unknown. Using horned Onthophagus beetles, we investigated the mechanisms that instruct the development of the dorsal adult head and the formation and integration of head horns, one of the most elaborate classes of secondary sexual weapons in the animal kingdom. RESULTS: Using region-specific RNAseq and gene knockdowns, we (i) show that the head is compartmentalized along multiple axes, (ii) identify striking parallels between morphological and transcriptional complexity across regions, yet (iii) fail to identify a horn-forming gene module. Instead, (iv) our results support that sex-biased regulation of a shared transcriptional repertoire underpins the formation of horned and hornless heads. Furthermore, (v) we show that embryonic head patterning genes frequently maintain expression within the dorsal head well into late post-embryonic development, thereby possibly facilitating the repurposing of such genes within novel developmental contexts. Lastly, (vi) we identify novel functions for several genes including three embryonic head patterning genes in the integration of both posterior and anterior head horns. CONCLUSIONS: Our results illuminate how the adult insect head is patterned and suggest mechanisms capable of integrating novel traits within ancient trait complexes in a sex- and species-specific manner. More generally, our work underscores how significant morphological innovation in developmental evolution need not require the recruitment of new genes, pathways, or gene networks but instead may be scaffolded by pre-existing developmental machinery.

Adenohypophysis placodal precursors exhibit distinctive features within the rostral preplacodal ectoderm.

Placodes are discrete thickenings of the vertebrate cranial ectoderm that generate morpho-functionally distinct structures, such as the adenohypophysis, olfactory epithelium and lens. All placodes arise from a horseshoe-shaped preplacodal ectoderm in which the precursors of individual placodes are intermingled. However, fate-map studies indicated that cells positioned at the preplacodal midline give rise to only the adenohypophyseal placode, suggesting a unique organization of these precursors within the preplacode. To test this possibility, we combined embryological and molecular approaches in chick embryos to show that, at gastrula stage, adenohypophyseal precursors are clustered in the median preplacodal ectoderm, largely segregated from those of the adjacent olfactory placode. Median precursors are elongated, densely packed and, at neurula stage, express a molecular signature that distinguishes them from the remaining preplacodal cells. Olfactory placode precursors and midline neural cells can replace ablated adenohypophyseal precursors up to head-fold stage, although with a more plastic organization. We thus propose that adenohypophyseal placode precursors are unique within the preplacodal ectoderm possibly because they originate the only single placode and the only one with an endocrine character.

Dlx1/2 are Central and Essential Components in the Transcriptional Code for Generating Olfactory Bulb Interneurons.

Generation of olfactory bulb (OB) interneurons requires neural stem/progenitor cell specification, proliferation, differentiation, and young interneuron migration and maturation. Here, we show that the homeobox transcription factors Dlx1/2 are central and essential components in the transcriptional code for generating OB interneurons. In Dlx1/2 constitutive null mutants, the differentiation of GSX2+ and ASCL1+ neural stem/progenitor cells in the dorsal lateral ganglionic eminence is blocked, resulting in a failure of OB interneuron generation. In Dlx1/2 conditional mutants (hGFAP-Cre; Dlx1/2F/- mice), GSX2+ and ASCL1+ neural stem/progenitor cells in the postnatal subventricular zone also fail to differentiate into OB interneurons. In contrast, overexpression of Dlx1&2 in embryonic mouse cortex led to ectopic production of OB-like interneurons that expressed Gad1, Sp8, Sp9, Arx, Pbx3, Etv1, Tshz1, and Prokr2. Pax6 mutants generate cortical ectopia with OB-like interneurons, but do not do so in compound Pax6; Dlx1/2 mutants. We propose that DLX1/2 promote OB interneuron development mainly through activating the expression of Sp8/9, which further promote Tshz1 and Prokr2 expression. Based on this study, in combination with earlier ones, we propose a transcriptional network for the process of OB interneuron development.

Molecular control of two novel migratory paths for CGE-derived interneurons in the developing mouse brain.

GABAergic interneurons are highly heterogeneous and originate in the subpallium mainly from the medial (MGE) and caudal (CGE) ganglionic eminences according to a precise temporal sequence. MGE-derived cells disperse dorsally and migrate towards all regions of the cortex, but little is known about how CGE-derived cells reach their targets during development. Here, we unravel the existence of two novel CGE caudo-rostral migratory streams, one located laterally (LMS) and the other one more medially (MMS), that, together with the well-known caudal migratory stream (CMS), contribute to populate the neocortex, hippocampus and amygdala. These paths appear in a precise temporal sequence and express a distinct combination of transcription factors, such as SP8, PROX1, COUP-TFI and COUP-TFII. By inactivating COUP-TFI in developing interneurons, the lateral and medial streams are perturbed and expression of SP8 and COUP-TFII affected. As a consequence, adult mutant neocortices have laminar-specific alterations of distinct cortical interneuron subtypes. Overall, we propose that the existence of spatially and temporally regulated migratory paths in the subpallium contributes to the laminar distribution and specification of distinct interneuron subpopulations in the adult brain.

Holoprosencephaly, orofacial cleft, and frontonaso-orbital encephaloceles: Genetic evaluation of a possible new syndrome.

Here we report on a Brazilian child who presented semilobar holoprosencephaly, frontonasal encephaloceles and bilateral cleft lip and palate. Malformations also included agenesis of the corpus callosum, abnormal cortical gyres, dilation of the aqueduct, bilateral endolymphatic sac, bilateral cystic cocci-vestibular malformation, and a cribriform defect. The 3D TC craniofacial images showed abnormal frontonasal transition region, with a bone bifurcation, and partial agenesis of nasal bone. The trunk and upper and lower limbs were normal. To our knowledge, this rare association of holoprocensephaly with frontonaso-orbital encephaloceles without limb anomalies has never been reported before. Karyotype was normal. SNP-array showed no copy-number alterations but revealed 25% of regions of homozygosity (ROH) with normal copy number, indicating a high coefficient of inbreeding, which significantly increases the risk for an autosomal recessive disorder. Whole exome sequencing analysis did not reveal any pathogenic or likely pathogenic variants. We discuss the possible influence of two variants of uncertain significance found within the patient's ROHs. First, a missense p.(Gly394Ser) in PCSK9, a gene involved in the regulation of plasma low-density lipoprotein cholesterol. Second, an inframe duplication p.(Ala75_Ala81dup) in SP8, a zinc-finger transcription factor that regulates signaling centers during craniofacial development. Further studies and/or the identification of other patients with a similar phenotype will help elucidate the genetic etiology of this complex case.

Sp5 and Sp8 recruit ß-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription.

The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factors Sp5 and Sp8 (i.e., Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/ß-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/ß-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of ß-catenin to target gene enhancers. Because Sp5 is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/ß-catenin pathway-specific transcript on factor that functions in a feed-forward loop to robustly activate select Wnt target genes.

UTR-specific knockdown of Distal-less and Sp8 leads to new phenotypic variants in the flour beetle Tribolium.

RNA interference (RNAi)-mediated knockdown serves as an effective technique for the functional analysis of developmental genes that is well established in many organisms. In the beetle Tribolium castaneum, double-stranded RNA is applied by simple injection and distributes systemically within the tissue. Thus, systematic testing for RNAi specificity and efficiency is easily possible in this organism. Generally, the use of non-overlapping dsRNA fragments yielding qualitatively identical phenotypes is the method of choice to verify target-specific knockdown effects. Here, we show that UTR-specific RNAi results in different effects regarding quality, severity and penetrance when compared to RNAi fragments directed at the coding region. Furthermore, when using 3'UTR-specific dsRNA, we first describe the Distal-lessRNAi antenna-to-leg transformation phenotype in the Tribolium larva, which has only been observed in the adult beetle and Drosophila so far. In addition, we unexpectedly observed sterility effects caused by 3'UTR-specific knockdown of the Tribolium-Sp8 orthologue that is not seen when dsRNA targeted a sequence within the coding-region or the 5'UTR that itself led to early embryonic lethality. We conclude that targeting UTR sequences by region-specific RNAi can reveal unexpected new aspects of gene function applicable in basic research and crop protection.

The Transcription Factors COUP-TFI and COUP-TFII have Distinct Roles in Arealisation and GABAergic Interneuron Specification in the Early Human Fetal Telencephalon.

In human telencephalon at 8-12 postconceptional weeks, ribonucleic acid quantitative sequencing and immunohistochemistry revealed cortical chicken ovalbumin upstream promotor-transcription factor 1 (COUP-TFI) expression in a high ventro-posterior to low anterior gradient except for raised immunoreactivity in the anterior ventral pallium. Unlike in mouse, COUP-TFI and SP8 were extensively co-expressed in dorsal sensory neocortex and dorsal hippocampus whereas COUPTFI/COUPTFII co-expression defined ventral temporal cortex and ventral hippocampus. In the ganglionic eminences (GEs) COUP-TFI immunoreactivity demarcated the proliferative zones of caudal GE (CGE), dorsal medial GE (MGE), MGE/lateral GE (LGE) boundary, and ventral LGE whereas COUP-TFII was limited to ventral CGE and the MGE/LGE boundary. Co-labeling with gamma amino butyric acidergic interneuron markers revealed that COUP-TFI was expressed in subpopulations of either MGE-derived (SOX6+) or CGE-derived (calretinin+/SP8+) interneurons. COUP-TFII was mainly confined to CGE-derived interneurons. Twice as many GAD67+ cortical cells co-labeled for COUP-TFI than for COUP-TFII. A fifth of COUP-TFI cells also co-expressed COUP-TFII, and cells expressing either transcription factor followed posterior or anterio-lateral pathways into the cortex, therefore, a segregation of migration pathways according to COUP-TF expression as proposed in mouse was not observed. In cultures differentiated from isolated human cortical progenitors, many cells expressed either COUP-TF and 30% also co-expressed GABA, however no cells expressed NKX2.1. This suggests interneurons could be generated intracortically from progenitors expressing either COUP-TF.

Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary in the spinal cord.

Progenitor cells are segregated into multiple domains along the dorsoventral axis of the vertebrate neural tube, and each progenitor domain generates particular types of neurons. Selective cross-repressive interactions between pairs of class I and class II transcription factors play important roles in patterning neural progenitors into domains with clear boundaries. Here, we provide evidence that the zinc-finger protein Sp8 plays a supplementary role to Pax6 in establishing the pMN/p3 domain boundary through mutually repressive interactions with the class II protein Nkx2-2. The ventral limit of Sp8 expression is complementary to the dorsal limit of Nkx2-2 expression at the pMN/p3 boundary. Sp8 and Nkx2-2 exert cross-repressive interactions, and changing the expression of Sp8 and Nkx2-2 is coupled with pMN and p3 progenitor fate conversion. Sp8 exerts its neural patterning activities by acting as a transcriptional activator. The expression of a repressive form of Sp8 results in the selective inhibition of motor neuron generation and the ectopic induction of Nkx2-2 expression. Sp8 expression is positively regulated by, but not completely dependent on, Pax6. Furthermore, whereas loss of Pax6 function alone results in disruption of the pMN/p3 domain boundary only in the rostral levels of the spinal cord, loss of both Sp8 and Pax6 functions results in disruption of the pMN/p3 domain boundary along the whole rostrocaudal axis of the spinal cord. We conclude that Sp8 plays a supplementary role to Pax6 in specifying the pMN over p3 progenitor fate through cross-repressive interactions with Nkx2-2.

Novel regulators of spermatogenesis.

Spermatogenesis is a multistep process that supports the production of millions of sperm daily. Understanding of the molecular mechanisms that regulate spermatogenesis has been a major focus for decades. Yet, the regulators involved in different cellular processes of spermatogenesis remain largely unknown. Human diseases that result in defective spermatogenesis have provided hints on the molecular mechanisms regulating this process. In this review, we have summarized recent findings on the function and signaling mechanisms of several genes that are known to be associated with disease or pathological processes, including CFTR, CD147, YWK-II and CT genes, and discuss their potential roles in regulating different processes of spermatogenesis.

Human and monkey striatal interneurons are derived from the medial ganglionic eminence but not from the adult subventricular zone.

In adult rodent and monkey brains, newly born neurons in the subventricular zone (SVZ) in the wall of the lateral ventricle migrate into the olfactory bulb (OB) via the rostral migratory stream (RMS). A recent study reported that interneurons are constantly generating in the adult human striatum from the SVZ. In contrast, by taking advantage of the continuous expression of Sp8 from the neuroblast stage through differentiation into mature interneurons, we found that the adult human SVZ does not generate new interneurons for the striatum. In the adult human SVZ and RMS, very few neuroblasts were observed, and most of them expressed the transcription factor Sp8. Neuroblasts in the adult rhesus monkey SVZ-RMS-OB pathway also expressed Sp8. In addition, we observed that Sp8 was expressed by most adult human and monkey OB interneurons. However, very few Sp8+ cells were in the adult human striatum. This suggests that neuroblasts in the adult human SVZ and RMS are likely destined for the OB, but not for the striatum. BrdU-labeling results also revealed few if any newly born neurons in the adult rhesus monkey striatum. Finally, on the basis of transcription factor expression, we provide strong evidence that the vast majority of interneurons in the human and monkey striatum are generated from the medial ganglionic eminence during embryonic developmental stages, as they are in rodents. We conclude that, although a small number of neuroblasts exist in the adult human SVZ, they do not migrate into the striatum and become mature striatal interneurons.

Sp8 and COUP-TF1 reciprocally regulate patterning and Fgf signaling in cortical progenitors.

To gain new insights into the transcriptional regulation of cortical development, we examined the role of the transcription factor Sp8, which is downstream of Fgf8 signaling and known to promote rostral cortical development. We have used a binary transgenic system to express Sp8 throughout the mouse telencephalon in a temporally restricted manner. Our results show that misexpression of Sp8 throughout the telencephalon, at early but not late embryonic stages, results in cortical hypoplasia, which is accompanied by increased cell death, reduced proliferation, and precocious neuronal differentiation. Misexpression of Sp8 at early developmental stages represses COUP-TF1 expression, a negative effector of Fgf signaling and a key promoter of posterior cortical identity, while ablation of Sp8 has the opposite effect. In addition, transgenic misexpression of COUP-TF1 resulted in downregulation of Sp8, indicating a reciprocal cross-regulation between these 2 transcription factors. Although Sp8 has been suggested to induce and/or maintain Fgf8 expression in the embryonic telencephalon, neither Fgf8 nor Fgf15 was upregulated using our gain-of-function approach. However, misexpression of Sp8 greatly increased the expression of Fgf target molecules, suggesting enhanced Fgf signaling. Thus, we propose that Sp8 promotes rostral and dorsomedial cortical development by repressing COUP-TF1 and promoting Fgf signaling in pallial progenitors.

SP8 regulates signaling centers during craniofacial development.

Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations.

Comprehensive insights into the mechanism of keratin degradation and exploitation of keratinase to enhance the bioaccessibility of soybean protein.

Keratin is a recalcitrant protein and can be decomposed in nature. However, the mechanism of keratin degradation is still not well understood. In this study, Bacillus sp. 8A6 can completely degrade the feather in 20 h, which is an efficient keratin degrader reported so far. Comprehensive transcriptome analysis continuously tracks the metabolism of Bacillus sp. 8A6 throughout its growth in feather medium. It reveals for the first time how the strain can acquire nutrients and energy in an oligotrophic feather medium for proliferation in the early stage. Then, the degradation of the outer lipid layer of feather can expose the internal keratin structure for disulfide bonds reduction by sulfite from the newly identified sulfite metabolic pathway, disulfide reductases and iron uptake. The resulting weakened keratin has been further proposedly de-assembled by the S9 protease and hydrolyzed by synergistic effects of the endo, exo and oligo-proteases from S1, S8, M3, M14, M20, M24, M42, M84 and T3 families. Finally, bioaccessible peptides and amino acids are generated and transported for strain growth. The keratinase has been applied for soybean hydrolysis, which generates 2234 peptides and 559.93 mg/L17 amino acids. Therefore, the keratinases, inducing from the poultry waste, have great potential to be further applied for producing bioaccessible peptides and amino acids for feed industry.

Regulation of the Human IL-10RB Gene Expression by Sp8 and Sp9.

BACKGROUND: Interleukin-10 (IL-10) is a classic anti-inflammatory cytokine that exerts its effects via the receptor complexes IL-10RA and IL-10RB. Loss of IL-10RB results in many diseases. Moreover, IL-10RB is closely associated with neuronal survival and synaptic formation. However, the regulation of IL-10RB gene expression remains elusive. OBJECTIVE: To investigate whether the expression of IL-10RB gene is increased in brain of Alzheimer's disease (AD) and its transcriptional regulation. METHODS: We examined the gene expression of AD patient brain from public database and detected the protein expression of AD model mouse brain by western blot. We constructed a variety of reporter gene plasmids with different lengths or mutation sites, tested the promoter activity and defined the functional region of the promoter with the luciferase reporter assay. The protein-DNA binding between transcription factors and the promoter was analyzed using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). RESULTS: We found that the IL-10RB is elevated in the brain of AD patient and AD model mice. The minimal promoter of the IL-10RB gene is located in the -90 to +51 bp region (relative to the transcriptional start site) and is sufficient for high-level expression of the IL-10RB gene. Transcription factors Sp8 and Sp9 bind to the IL-10RB promoter in vitro. The overexpression or knockdown of Sp8 and Sp9 affected the IL-10RB promoter activity and its gene expression. CONCLUSION: Our study functionally characterized the promoter of the IL-10RB gene and demonstrated that Sp8 and Sp9 regulated its expression.

Atto 465 Derivative Is a Nuclear Stain with Unique Excitation and Emission Spectra Useful for Multiplex Immunofluorescence Histochemistry.

Multiplex immunofluorescence (mIF) is an effective technique for the maximal visualization of multiple target proteins in situ. This powerful tool is mainly limited by the spectral overlap of the currently available synthetic fluorescent dyes. The fluorescence excitation wavelengths ranging between 405 and 488 nm are rarely used in mIF imaging and serve as a logical additional slot for a fluorescent probe. In the present study, we demonstrate that the addition of 2,3,4,5,6-pentafluoroaniline to Atto 465 NHS ester, creating Atto 465-pentafluoroaniline (Atto 465-p), generates a bright nuclear stain in the violet-blue region of the visible spectrum. This allows the 405 nm excitation and emission, classically used for nuclear counterstains, to be used for the detection of another target protein. This increases the flexibility of the mIF panel and, with appropriate staining and microscopy, enables the quantitative analysis of at least six targets in one tissue section. (J Histochem Cytochem XX: XXX-XXX, XXXX).

SP8 Promotes an Aggressive Phenotype in Hepatoblastoma via FGF8 Activation.

Hepatoblastoma (HB) is the most common malignant liver tumor in childhood and it generally has a good prognosis. However, if associated with aggressive metastatic disease, outcome is still poor. The molecular mechanisms leading to metastatic spread in HB patients are still unknown. By combining RNA-sequencing and a genome-wide methylome analysis, we identified the transcription factor SP8 and the growth factor FGF8 among the most strongly upregulated genes in metastatic HB cases, with a concomitant robust demethylation of the respective promoter regions. Of note, high expression of both candidates was associated with the aggressive C2 subtype of the 16-gene signature and poor survival. Chromatin immunoprecipitation revealed a direct transcriptional regulation of FGF8 through binding of SP8 to the FGF8 promoter. Gain- and loss-of-function experiments proved promoting effects of SP8 on motility, self-renewal, migration, and the invasive potential of HB cells. Moreover, stable overexpression of SP8 in Hep3B cells resulted in the acquisition of a mesenchymal phenotype and a strong upregulation of epithelial-mesenchymal transition-associated genes. Using KRAB-mediated CRISPR-dCas9 interference directed against FGF8, we could show that FGF8 is essential for the SP8-mediated aggressive tumor behavior. Treatment of HB cell lines with the pan SP family inhibitor mithramycin A resulted in a significant inhibition of their clonogenic growth. In summary, we identified SP8 and FGF8 as key players in aggressive traits of HB and propose SP8 inhibiting drugs as a new effective treatment strategy especially for metastatic tumors.