David Jukam

Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes

The mammalian vomeronasal organ (VNO), a part of the olfactory system, detects pheromones—chemical signals that modulate social and reproductive behaviours. But the molecular receptors in the VNO that detect these chemosensory stimuli remain undefined. Candidate pheromone receptors are encoded by two distinct and complex superfamilies of genes, V1r and V2r (refs 3 and 4), which code for receptors with seven transmembrane domains. These genes are selectively expressed in sensory neurons of the VNO. However, there is at present no functional evidence for a role of these genes in pheromone responses. Here, using chromosome engineering technology, we delete in the germ line of mice a ∼600-kilobase genomic region that contains a cluster of 16 intact V1r genes. These genes comprise two of the 12 described V1r gene families, and represent ∼12% of the V1r repertoire. The mutant mice display deficits in a subset of VNO-dependent behaviours: the expression of male sexual behaviour and maternal aggression is substantially altered. Electrophysiologically, the epithelium of the VNO of such mice does not respond detectably to specific pheromonal ligands. The behavioural impairment and chemosensory deficit support a role of V1r receptors as pheromone receptors.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

Significance Embryos depend on maternally deposited RNA until zygotic transcription activates. In many species, genome activation coincides with cell cycle lengthening and cellular motility, which collectively comprise the midblastula transition (MBT). A long-standing model is that MBT onset is controlled by titration of a maternally loaded inhibitor against exponentially increasing DNA. To identify MBT inhibitors, we developed an assay using Xenopus egg extract that recapitulates transcriptional activation only above a DNA-to-cytoplasm ratio similar to MBT embryos and identified histones H3 and H4 as inhibitors. Changing histone levels quantitatively shifts the DNA concentration required for transcription in vitro and alters the onset of both zygotic transcription and cell cycle lengthening in vivo. Our work strongly supports histones as a titrated MBT inhibitor. During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

Opposite Feedbacks in the Hippo Pathway for Growth Control and Neural Fate

Introduction: A finite number of signaling pathways are repurposed during animal development to regulate an extraordinary array of cellular decisions. Elucidating context-specific mechanisms is crucial for understanding how cellular diversity is generated and for defining potential avenues of pathway misregulation during disease. The Hippo tumor suppressor pathway has been primarily studied in growth control where it inhibits the oncogenic transcriptional coactivator Yorkie (Yki) (YAP/TAZ in vertebrates). The Hippo pathway also functions in nongrowth contexts such as postmitotic fate specification. In the Drosophila visual system, R8 photoreceptor neurons terminally differentiate into one of two alternative subtypes that express either blue-light–sensitive Rhodopsin5 (Rh5) or green-light–sensitive Rhodopsin6 (Rh6). These mutually exclusive cell fates are established by the Hippo pathway kinase warts and the growth regulator gene melted, which repress each other’s expression. However, the mechanisms underlying the context-specific use of the Hippo pathway in postmitotic fate decisions remain unclear. Context-specific regulation by the Hippo signaling in postmitotic photoreceptors. The Hippo pathway uses negative feedback through its transcriptional effector Yki for homeostatic control of proliferation. In Drosophila eyes, two alternative fates of blue- versus green-sensitive R8 photoreceptors are regulated by antagonism between the growth regulator Melted and the Hippo pathway. Contrary to the growth mechanism, Yki positive feedback and a cell-type–restricted transcription factor network promote repurposing of the Hippo pathway for binary fate decisions. Methods: To define the regulatory mechanisms of Hippo-dependent cell fate decisions in Drosophila photoreceptor neurons, we used a combination of genetic epistasis analyses, in vivo cis-regulatory studies, a candidate gene RNA interference screen, and cell culture–based transcription assays Results: We show that the transcriptional output of the Hippo pathway in photoreceptor differentiation, as in cell proliferation, is mediated through the factors Yki and Scalloped. In contrast to growth control, where Yki limits its own activity by negative feedback, we identify two Yki positive-feedback mechanisms: In blue-sensitive Rh5 photoreceptors, Yki represses its own negative regulator warts, downstream of melted; Yki also promotes melted expression, which subsequently represses warts to further promote Yki function. Yki cooperates with the transcription factors Orthodenticle (Otd) and Traffic Jam (Tj) to promote melted expression and Rh5 photoreceptor fate. Otd and Tj, othologs of the mammalian OTX/CRX and MAF/NRL transcription factors, form an evolutionarily conserved transcriptional module for generating photoreceptor subtype diversity. We also show that the transcription factors Senseless and Pph13 create a permissive environment for Warts/Hippo signaling to promote the alternative “default” green-sensitive Rh6 fate. Hence, Hippo pathway function integrates with four cell-type–restricted transcription factors, each promoting genetically different aspects of R8 subtypes, such that Yki activity ultimately coordinates the binary fate decision between blue- and green-sensitive photoreceptors. Discussion: This work illustrates how molecular signaling pathways can adopt context-specific regulation. Yki positive feedback in the photoreceptor fate decision is opposite to the negative feedback found in Hippo growth control. These distinct network-level feedback mechanisms provide context-appropriate functions: homeostasis to fine-tune growth regulation and an all-or-nothing fate decision to ensure robust differentiation of sensory neuron subtypes. Altering network-level systems properties, such as positive or negative feedback, within biochemically conserved pathways may be broadly used to co-opt signaling networks for use in cellular contexts as distinct as proliferation and terminal differentiation. Complexity and Diversity Complex organisms must produce and maintain an extraordinary diversity of cell and tissue types with a limited number of genes and molecular pathways. Cells accomplish this by reusing the same signaling networks at different times, in different tissues, and for different purposes, yet how this context-specificity is achieved is poorly understood. Jukam et al. (1238016, published online 29 August) show how a set of genes that function in cell and tissue growth can be used again in nondividing fly photoreceptor neurons to ensure that flies develop appropriate sensitivity to both blue and green light. The Hippo pathway undergoes a regulatory change—from negative to positive feedback—that requires a tissue-specific transcription factor network. This network uses evolutionarily conserved regulatory factors whose mutations in humans result in degenerative retinal diseases. The context-appropriate positive feedback in flies ensures an all-or-nothing fate decision necessary to establish a functional visual system. Hippo directs cell differentiation and fate through context- and tissue-specific feedback and transcription networks. Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network—including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module—that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.

Binary Regulation of Hippo Pathway by Merlin/NF2, Kibra, Lgl, and Melted Specifies and Maintains Postmitotic Neuronal Fate

Patterning the Drosophila retina for color vision relies on postmitotic specification of photoreceptor subtypes. R8 photoreceptors express one of two light-sensing Rhodopsins, Rh5 or Rh6. This fate decision involves a bistable feedback loop between Melted, a PH-domain protein, and Warts, a kinase in the Hippo growth pathway. Here, we show that a subset of the Hippo pathway-Merlin, Kibra, and Lethal(2)giant larvae (Lgl), but not Expanded or Fat-is required for Warts expression and activity in R8 to specify Rh6 fate. Melted represses warts transcription to disrupt Hippo pathway activity and specify Rh5 fate. Therefore, R8 Hippo signaling exhibits ON-or-OFF regulation, promoting mutually exclusive fates. Furthermore, Merlin and Lgl are continuously required to maintain R8 neuronal subtypes. These results reveal roles for Merlin, Kibra, and Lgl in neuronal specification and maintenance and show that the Hippo pathway is reimplemented for sensory neuron fate by combining canonical and noncanonical regulatory steps.

Binary Fate Decisions in Differentiating Neurons

Neural cell fate programs must generate an enormous number of neurons with distinct adult functions. The decision to choose one neuronal subtype from two alternatives--a binary fate decision--is one way to diversify neuronal subtypes during nervous system development. Recent progress has been made in describing the genetic programs that define late-stage neuronal identity. Here, we review mechanisms that control how such fate decisions generate two different postmitotic, terminally differentiated neuronal subtypes. We survey examples from Caenorhabditis elegans and Drosophila that demonstrate different modes of binary neuronal fate specification that depend on cell division, lineage, stochastic gene expression, or extracellular signals. Comparison of these strategies reveals that, although organisms use diverse approaches to generate neural diversity, some common themes do exist.

Regional Modulation of a Stochastically Expressed Factor Determines Photoreceptor Subtypes in the Drosophila Retina

Stochastic mechanisms are sometimes utilized to diversify cell fates, especially in nervous systems. In the Drosophila retina, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) controls photoreceptor subtype choice. In one randomly distributed subset of R7 photoreceptors, Ss activates Rhodopsin4 (Rh4) and represses Rhodopsin3 (Rh3); counterparts lacking Ss express Rh3 and repress Rh4. In the dorsal third region of the retina, the Iroquois Complex transcription factors induce Rh3 in Rh4-expressing R7s. Here, we show that Ss levels are controlled in a binary on/off manner throughout the retina yet are attenuated in the dorsal third region to allow Rh3 coexpression with Rh4. Whereas the sensitivity of rh3 repression to differences in Ss levels generates stochastic and regionalized patterns, the robustness of rh4 activation ensures its stochastic expression throughout the retina. Our findings show how stochastic and regional inputs are integrated to control photoreceptor subtype specification in the Drosophila retina.

Zygotic Genome Activation in Vertebrates

The first major developmental transition in vertebrate embryos is the maternal-to-zygotic transition (MZT) when maternal mRNAs are degraded and zygotic transcription begins. During the MZT, the embryo takes charge of gene expression to control cell differentiation and further development. This spectacular organismal transition requires nuclear reprogramming and the initiation of RNAPII at thousands of promoters. Zygotic genome activation (ZGA) is mechanistically coordinated with other embryonic events, including changes in the cell cycle, chromatin state, and nuclear-to-cytoplasmic component ratios. Here, we review progress in understanding vertebrate ZGA dynamics in frogs, fish, mice, and humans to explore differences and emphasize common features.

Single-base pair differences in a shared motif determine differential Rhodopsin expression

Broad versus restricted expression Color vision in fruit flies requires the restricted expression of light-sensing rhodopsins with different wavelength sensitivities in subsets of photoreceptors. However, all photoreceptors express factors that transduce and amplify the visual signal. Rister et al. found that the distinct expression patterns are determined by a highly tunable regulatory motif. Genes that are broadly expressed have a palindromic variant of the motif. Spatially restricted rhodopsin genes display single-base-pair changes that alter the symmetry of the palindrome and are critical for subtype-specific expression. These findings on the differential regulation of gene expression in fly photoreceptors have implications for the evolution of neuronal subtype diversity. Science, this issue p. 1258 Single–base pair changes in a transcription factor binding motif modulate cell type–specific gene expression. The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11–base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli.

The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted. Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein. Summary: During cell fate specification in the Drosophila eye, BEAF-32 regulates expression of Hippo pathway components and acts downstream of Warts to control photoreceptor identity.

Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.