Retinal Determination Gene Research Articles

Genetic mechanism regulating diversity in the placement of eyes on the head of animals

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Vestigial organs alter fossil placements in an ancient group of terrestrial chelicerates

Vestigial organs provide a link between ancient and modern traits and therefore have great potential to resolve the phylogeny of contentious fossils that bear features not seen in extant species. Here we show that extant daddy-longlegs (Arachnida, Opiliones), a group once thought to possess only one pair of eyes, in fact additionally retain a pair of vestigial median eyes and a pair of vestigial lateral eyes. Neuroanatomical gene expression surveys of eye-patterning transcription factors, opsins, and other structural proteins in the daddy-longlegs Phalangium opilio show that the vestigial median and lateral eyes innervate regions of the brain positionally homologous to the median and lateral eye neuropils, respectively, of chelicerate groups like spiders and horseshoe crabs. Gene silencing of eyes absent shows that the vestigial eyes are under the control of the retinal determination gene network. Gene silencing of dachshund disrupts the lateral eyes, but not the median eyes, paralleling loss-of-function phenotypes in insect models. The existence of lateral eyes in extant daddy-longlegs bears upon the placement of the oldest harvestmen fossils, a putative stem group that possessed both a pair of median eyes and a pair of lateral eyes. Phylogenetic analysis of harvestman relationships with an updated understanding of lateral eye incidence resolved the four-eyed fossil group as a member of the extant daddy-longlegs suborder, which in turn resulted in older estimated ages of harvestman diversification. This work underscores that developmental vestiges in extant taxa can influence our understanding of character evolution, placement of fossils, and inference of divergence times.

Regulation of Eye Determination and Regionalization in the Spider Parasteatoda tepidariorum.

Animal visual systems are enormously diverse, but their development appears to be controlled by a set of conserved retinal determination genes (RDGs). Spiders are particular masters of visual system innovation, and offer an excellent opportunity to study the evolution of animal eyes. Several RDGs have been identified in spider eye primordia, but their interactions and regulation remain unclear. From our knowledge of RDG network regulation in Drosophila melanogaster, we hypothesize that orthologs of Pax6, eyegone, Wnt genes, hh, dpp, and atonal could play important roles in controlling eye development in spiders. We analyzed the expression of these genes in developing embryos of the spider Parasteatoda tepidariorum, both independently and in relation to the eye primordia, marked using probes for the RDG sine oculis. Our results support conserved roles for Wnt genes in restricting the size and position of the eye field, as well as for atonal initiating photoreceptor differentiation. However, we found no strong evidence for an upstream role of Pax6 in eye development, despite its label as a master regulator of animal eye development; nor do eyg, hh or dpp compensate for the absence of Pax6. Conversely, our results indicate that hh may work with Wnt signaling to restrict eye growth, a role similar to that of Sonic hedgehog (Shh) in vertebrates.

Systemic paralogy and function of retinal determination network homologs in arachnids

BackgroundArachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, duplication of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models.ResultsWe investigated a sister species pair of Israeli cave whip spiders, Charinus ioanniticus and C. israelensis (Arachnopulmonata, Amblypygi), of which one species has reduced eyes. We generated embryonic transcriptomes for both Amblypygi species, and discovered that several RDGN homologs exhibit duplications. We show that duplication of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types.ConclusionsOur work establishes a foundation to investigate the genetics of troglomorphic adaptations in cave arachnids, and links differential gene expression to an arthropod eye phenotype for the first time outside of Pancrustacea. Our results support the conservation of at least one RDGN component across Arthropoda and provide a framework for identifying the role of gene duplications in generating arachnid eye diversity.

An E3 ubiquitin ligase, cullin-4 regulates retinal differentiation in Drosophila eye.

During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.

RDGN-based predictive model for the prognosis of breast cancer

BackgroundBreast cancer is the most diagnosed malignancy in females in the United States. The members of retinal determination gene network (RDGN) including DACH, EYA, as well as SIX families participate in the proliferation, apoptosis, and metastasis of multiple tumors including breast cancer. A comprehensive predictive model of RDGN might be helpful to herald the prognosis of breast cancer patients.MethodsIn this study, the Gene Expression Ominibus (GEO) and Gene Set Expression Analysis (GSEA) algorithm were used to investigate the effect of RDGN members on downstream signaling pathways. Besides, based on The Cancer Genome Atlas (TCGA) database, we explored the expression patterns of RDGN members in tumors, normal tissues, and different breast cancer subtypes. Moreover, we estimated the relationship between RDGN members and the outcomes of breast cancer patients. Lastly, we constructed a RDGN-based predictive model by Cox proportional hazard regression and verified the model in two separate GEO datasets.ResultsThe results of GSEA showed that the expression of DACH1 was negatively correlated with cell cycle and DNA replication pathways. On the contrary, the levels of EYA2 and SIX1 were significantly positively correlated with DNA replication, mTOR, and Wnt pathways. Further investigation in TCGA database indicated that DACH1 expression was lower in breast cancers especially basal-like subtype. In the meanwhile, SIX1 was remarkably upregulated in breast cancers while EYA2 level was increased in Basal-like and Her-2 enriched subtypes. Survival analyses demonstrated that DACH1 was a favorable factor while EYA2 and SIX1 were risk factors for breast cancer patients. Given the results of Cox proportional hazard regression analysis, two members of RDGN were involved in the present predictive model and patients with high model index had poorer outcomes.ConclusionThis study showed that aberrant RDGN expression was an unfavorable factor for breast cancer. This RDGN-based comprehensively framework was meaningful for predicting the prognosis of breast cancer patients.

Dach1 regulates neural crest migration during embryonic development

Dachshund 1(Dach1) is a key component of the retinal determination gene network that plays significant roles in cell fate regulation. The vertebrate homolog of Drosophila dachshund has gained considerable importance as an essential regulator of development, but its functions during embryonic development remain elusive. We investigated the functional significance of dach1 during Xenopus embryogenesis using loss-of-function studies. Reverse transcription-polymerase chain reaction demonstrated the maternal nature of dach1, showing enhanced expression at the neurula stage of development, and morpholino oligonucleotide injection of dach1 induced phenotypic anomalies of microcephaly and reduced body length. Animal cap assays followed by whole-mount in-situ hybridization indicated the perturbed expression of neural and neural crest (NC) markers. Our data suggest the prerequisite functions of dach1 in NC migration during Xenopus embryogenesis. However, the developmental pathways regulated by dach1 during embryogenesis require further elucidation.

SIX1 Activates STAT3 Signaling to Promote the Proliferation of Thyroid Carcinoma via EYA1.

As a critical member of the Retinal Determination Gene Network (RDGN), SIX1 has been regarded as a tumor promoter in various types of cancer. However, its role in papillary thyroid carcinoma (PTC) has never been investigated. In this study, thyroid carcinoma tissue microarray staining was employed to identify the expression patterns of SIX1 and its co-activator EYA1. Papillary thyroid cancer cell lines, BCPAP, and TPC-1 cells were used to investigate the potential mechanism of SIX1 in vitro and in vivo. Flow cytometry analysis, MTT assay, the growth curve assay, colony formation assay, EdU incorporation and xenograft assay were performed to demonstrate the role of SIX1 in the malignant change of PTC cells. Western blot and Real-time PCR were used to detect the interaction among the SIX1, EYA1, and STAT3 signaling. In comparison with normal tissue, high expressions of SIX1 and EYA1 were associated with a malignant tumor. Importantly, SIX1 strongly correlated with EYA1 in thyroid carcinoma tissue microarray. Functional assays indicated SIX1 increased EYA1 expression by stabilizing EYA1 at the post-transcriptional level. Besides, SIX1 promoted the proliferation and invasion of thyroid carcinoma via activation of STAT3 signaling and its downstream targets in an EYA1-dependent manner. SIX1 can integrate with EYA1 to contribute to PTC development via activation of the classical STAT3 signaling. These data suggested targeting the abnormal activation of the SIX1/EYA1 complex may represent a novel therapeutic strategy for advanced PTC patients.

The regulation of cytokine signaling by retinal determination gene network pathway in cancer.

Tumor environment plays a pivotal role in determining cancer biology characteristics. Cytokine factors, as a critical component in tumor milieu, execute distinct functions in the process of tumorigenesis and progression via the autocrine or paracrine manner. The retinal determination gene network (RDGN), which mainly comprised DACH, SIX, and EYA family members, is required for the organ development in mammalian species. While the aberrant expression of RDGN is involved in the proliferation, apoptosis, angiogenesis, and metastasis of tumors via interacting with different cytokine-related signals, such as CXCL8, IL-6, TGF-β, FGF, and VEGF, in a cell- or tissue-dependent manner. Thus, joint detection of this pathway might be used as a potential biomarker for the stratification of target therapy and for the precision prediction of the prognosis of cancer patients.

A soy protein Lunasin can ameliorate amyloid-beta 42 mediated neurodegeneration in Drosophila eye

Alzheimer’s disease (AD), a fatal progressive neurodegenerative disorder, also results from accumulation of amyloid-beta 42 (Aβ42) plaques. These Aβ42 plaques trigger oxidative stress, abnormal signaling, which results in neuronal death by unknown mechanism(s). We misexpress high levels of human Aβ42 in the differentiating retinal neurons of the Drosophila eye, which results in the Alzheimer’s like neuropathology. Using our transgenic model, we tested a soy-derived protein Lunasin (Lun) for a possible role in rescuing neurodegeneration in retinal neurons. Lunasin is known to have anti-cancer effect and reduces stress and inflammation. We show that misexpression of Lunasin by transgenic approach can rescue Aβ42 mediated neurodegeneration by blocking cell death in retinal neurons, and results in restoration of axonal targeting from retina to brain. Misexpression of Lunasin downregulates the highly conserved cJun-N-terminal Kinase (JNK) signaling pathway. Activation of JNK signaling can prevent neuroprotective role of Lunasin in Aβ42 mediated neurodegeneration. This neuroprotective function of Lunasin is not dependent on retinal determination gene cascade in the Drosophila eye, and is independent of Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways. Furthermore, Lunasin can significantly reduce mortality rate caused by misexpression of human Aβ42 in flies. Our studies identified the novel neuroprotective role of Lunasin peptide, a potential therapeutic agent that can ameliorate Aβ42 mediated neurodegeneration by downregulating JNK signaling.

Issue Information

COVER PHOTOGRAPH: Repeated, colorized scanning electron micrographs of the fruit fly, Drosophila melanogaster, in which a portion of the ventral head epidermis has been transformed into an ectopic eye by the forced expression of the retinal determination gene sine oculis. The ectopic eye sits just below the normal compound eye. Photo by Brandon P. Weasner and Justin P. Kumar; From: The Fly Eye: Through the Looking Glass; Justin Kumar, Developmental Dynamics 247:111–123.

Homeodomain-interacting protein kinase phosphorylates the Drosophila Paired box protein 6 (Pax6) homologues Twin of eyeless and Eyeless.

Homeodomain-interacting protein kinase (Hipk), the Drosophila homologue of mammalian HIPK2, plays several important roles in regulating differentiation, proliferation, apoptosis, and stress responses and acts as a mediator for signals of diverse pathways, such as Notch or Wingless signalling. The Paired box protein 6 (Pax6) has two Drosophila homologues, Twin of eyeless (Toy) and Eyeless (Ey). Both stand atop the retinal determination gene network (RDGN), which is essential for proper eye development in Drosophila. Here, we set Hipk and the master regulators Toy and Ey in an enzyme-substrate relationship. Furthermore, we prove a physical interaction between Toy and Hipk in vivo using bimolecular fluorescence complementation. Using in vitro kinase assays with different truncated Toy constructs and mutational analyses, we mapped four Hipk phosphorylation sites of Toy, one in the paired domain (Ser121 ) and three in the C-terminal transactivation domain of Toy (Thr395 , Ser410 and Thr452 ). The interaction and phosphorylation of the master regulator Toy by Hipk may be important for precise tuning of signalling within the RDGN and therefore for Drosophila eye development.

Mutations that impair Eyes absent tyrosine phosphatase activity in vitro reduce robustness of retinal determination gene network output in Drosophila.

A limited collection of signaling networks and transcriptional effectors directs the full spectrum of cellular behaviors that comprise development. One mechanism to diversify regulatory potential is to combine multiple biochemical activities into the same protein. Exemplifying this principle of modularity, Eyes absent (Eya), originally identified as a transcriptional co-activator within the retinal determination gene network (RDGN), also harbors tyrosine and threonine phosphatase activities. Although mounting evidence argues for the importance of Eya’s phosphatase activities to mammalian biology, genetic rescue experiments in Drosophila have shown that the tyrosine phosphatase function is dispensable for normal development. In this study, we repeated these rescue experiments in genetically sensitized backgrounds in which the dose of one or more RDGN factor was reduced. Heterozygosity for sine oculis or dachshund, both core RDGN members, compromised the ability of phosphatase-dead eya, but not of the control wild type eya transgene, to rescue the retinal defects and reduced viability associated with eya loss. We speculate that Eya’s tyrosine phosphatase activity, although non-essential, confers robustness to RDGN output.

Eye Development in Sepia officinalis Embryo: What the Uncommon Gene Expression Profiles Tell Us about Eye Evolution.

In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including pax, six, eya, and dac playing also key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.

Dachshund Potentiates Hedgehog Signaling during Drosophila Retinogenesis.

Proper organ patterning depends on a tight coordination between cell proliferation and differentiation. The patterning of Drosophila retina occurs both very fast and with high precision. This process is driven by the dynamic changes in signaling activity of the conserved Hedgehog (Hh) pathway, which coordinates cell fate determination, cell cycle and tissue morphogenesis. Here we show that during Drosophila retinogenesis, the retinal determination gene dachshund (dac) is not only a target of the Hh signaling pathway, but is also a modulator of its activity. Using developmental genetics techniques, we demonstrate that dac enhances Hh signaling by promoting the accumulation of the Gli transcription factor Cubitus interruptus (Ci) parallel to or downstream of fused. In the absence of dac, all Hh-mediated events associated to the morphogenetic furrow are delayed. One of the consequences is that, posterior to the furrow, dac- cells cannot activate a Roadkill-Cullin3 negative feedback loop that attenuates Hh signaling and which is necessary for retinal cells to continue normal differentiation. Therefore, dac is part of an essential positive feedback loop in the Hh pathway, guaranteeing the speed and the accuracy of Drosophila retinogenesis.

Retinal determination genes coordinate neuroepithelial specification and neurogenesis modes in the Drosophila optic lobe.

Differences in neuroepithelial patterning and neurogenesis modes contribute to area-specific diversifications of neural circuits. In the Drosophila visual system, two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers, generate neuron subtypes for four ganglia in several ways. Whereas neuroepithelial cells in the medial OPC directly convert into neuroblasts, in an IPC subdomain they generate migratory progenitors by epithelial-mesenchymal transition that mature into neuroblasts in a second proliferative zone. The molecular mechanisms that regulate the identity of these neuroepithelia, including their neurogenesis modes, remain poorly understood. Analysis of Polycomblike revealed that loss of Polycomb group-mediated repression of the Hox gene Abdominal-B (Abd-B) caused the transformation of OPC to IPC neuroepithelial identity. This suggests that the neuroepithelial default state is IPC-like, whereas OPC identity is derived. Ectopic Abd-B blocks expression of the highly conserved retinal determination gene network members Eyes absent (Eya), Sine oculis (So) and Homothorax (Hth). These factors are essential for OPC specification and neurogenesis control. Finally, eya and so are also sufficient to confer OPC-like identity, and, in parallel with hth, the OPC-specific neurogenesis mode on the IPC.

Conditional knockout of retinal determination genes in differentiating cells in Drosophila.

Conditional gene knockout in postmitotic cells is a valuable technique which allows the study of gene function with spatiotemporal control. Surprisingly, in contrast to its long-term and extensive use in mouse studies, this technology is lacking in Drosophila. Here, we use a novel method for generating complete loss of eyes absent (eya) or sine oculis (so) function in postmitotic cells posterior to the morphogenetic furrow (MF). Specifically, genomic rescue constructs with flippase recognition target (FRT) sequences flanking essential exons are used to generate conditional null alleles. By removing gene function in differentiating cells, we show that eya and so are dispensable for larval photoreceptor differentiation, but are required for differentiation during pupal development. Both eya and so are necessary for photoreceptor survival and the apoptosis caused by loss of eya or so function is likely a secondary consequence of inappropriate differentiation. We also confirm their requirement for cone cell development and reveal a novel role in interommatidial bristle (IOB) formation. In addition, so is required for normal eye disc morphology. This is the first report of a knockout method to study eya and so function in postmitotic cells. This technology will open the door to a large array of new functional studies in virtually any tissue and at any stage of development or in adults.

The retinal determination gene network: from developmental regulator to cancer therapeutic target.

Although originally identified for its function in Drosophila melanogaster eye specification, the Retinal Determination Gene Network (RDGN) is essential for the development of multiple organs in mammals. The RDGN regulates proliferation, differentiation and autocrine signaling, and interacts with other key signaling pathways. Aberrant expression of RDGN members such as DACH, EYA and SIX contributes to tumor initiation and progression; indeed, the levels of RDGN members are clinically prognostic factors in various cancer types. Stimulation or suppression of the activities of these crucial components can block cancer cell proliferation, prevent cancer stem cell expansion and even reverse the EMT process, thereby attenuating malignant phenotypes. Thus, cancer therapeutic interventions targeting RDGN members should be pursued in future studies.

Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism's genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. The migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a 'coherent feed-forward' transcriptional subcircuit composed of Pax6, Six3, Six1/2, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

Endogenous Dach1 in cancer.

The Dachshund gene is a key component of the retinal determination gene network in Drosophila eye development. Recent studies have demonstrated an important role for the human Dachshund homologue 1 (DACH1) in tumorigenesis [1]. Mechanistic studies demonstrated that DACH1 regulates its target genes via either directly binding to specific DNA sequences within chromatin or indirectly through forming protein complex with other transcriptional factors, including c-Jun and Smad4. DACH1 restrained proliferation, migration in vitro and repressed tumor growth and metastasis in vivo. There prior observations were based on cultured cells engineering DACH1 expression. The physiological role of the endogenous DACH1 had not been determined in part because genetic deletion in the mice was periembryonic lethal. A recent study report in the May 15 issue of Cancer Research determined the role of endogenous Dach1 in the prostate using tissue specific deletion mice [2]. Using prostate cancer cell lines of distinct genetic background, DACH1 was shown to inhibit proliferation in vitro and tumor growth in vivo of both AR negative cancer cell (PC-3) and castration resistant AR positive cell (C4-2 and 22RV-1). Consistent with the prior finding that DACH1 inhibited cell cycle progression though inhibiting Cyclin D1 expression, conditional Dach1 knock out in the prostate by using Dach1 fl/fl/Probasin-Cre bi-transgenic increased the expression of cyclin D1, E and A, accompanied by enhanced DNA synthesis and reduced apoptosis [2]. Whole genomic expression profiling with functional pathway analysis identified the cytokine-cytokine receptor interaction as a key target of endogenous Dach1. CXCL family member CXCL-1, 2, 5, 6 and IL-6, 8 expressions were inhibited by DACH1 in PC-3 cell. DACH1 mRNA expression was reduced in metastatic human prostate cancer [3] and DACH1 abundance was inversely correlated with IL-6 and IL-8 (Fig. ​(Fig.1).1). In agreement, there was 1000 fold activation of IL-6 and IL-8 secretion when endogenous Dach1 was deleted in vivo from prostate epithelial cells (PEC). Functional assays, using immune neutralizing antibodies or purified recombinant cytokines to conditioned medium from wt or Dach1 KO PEC, proved that IL-6 and KC (homolog of IL-8 in mice) were the key downstream targets of Dach1 in governing cell migration. In addition, various single oncogene (c-Myc, NeuT, H-Ras or v-Src) transformed prostate epithelial cells (PEC) reduced Dach1 expression both in cultured cells and in extirpated tumor tissues [2]. Combined with previous finding that DACH1 represses ligand induced transcriptional activation of the androgen receptor and prostate cancer cellular proliferation in tissue culture [3], it is likely that DACH1 conveys distinct functions at different stages of prostate cancer onset and progression. Figure 1 Reduced expression of DACH1 activates heterotypic signaling in prostate cancer The transition from hormone-dependent to castrate-resistant prostate cancer (CRPC) is the major cause of therapeutic failure. Clinical studies have shown that increased IL-6 and IL-8 signaling correlates with CRPC and predicts poor prognosis [4]. IL-6 and IL-8 produced by prostate cancer cell promote cancer cell proliferation and invasion. Moreover, IL-8 signaling from tumor cells initiates cancer cell-stromal interaction to induce treatment resistance and angiogenesis [5]. The finding that DACH1 is a key endogenous gene that restrains cytokine signaling may have therapeutic relevance. The finding that DNA demethylation agents could restore DACH1 expression in PC-3 cells [2] suggests targeting DACH1 may be practical. DACH1 also directly suppressed IL-8 in breast cancer cells and inhibited KC-mediated lung metastasis [6]. A subsequent study in lung cancer has also demonstrated that DACH1 can suppress the secretion of CXCL5, thereby reducing CXCL5-mediated proliferation, migration and invasion. Moreover, as with prostate cancer, a reverse relationship between DACH1 and CXCL5 was reported in tumor samples and low DACH1 correlated with reduced survival in lung cancer patients [7]. Thus, DACH1 governs cell fate through intracellular transcriptional regulation and also through heterotypic signaling that determines cancer-stromal interaction, a key hallmark of cancer [8].