Esteban O. Mazzoni

Stochastic spineless expression creates the retinal mosaic for colour vision

Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called ‘pale’ and ‘yellow’, defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. Here we report that the Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless.

An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming.

Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

Keeping repressed genes repressed Hox genes confer positional identity to cells and tissues. Maintaining precise spatial patterns of Hox gene expression is vital during metazoan development. The transcriptional repressor CTCF is involved in the regulation of chromatin architecture. Narendra et al. show that a CTCF protein binding site insulates regions of active and repressed Hox gene expression from each other. This protects heterochromatin containing repressed Hox genes from the encroaching spread of active chromatin. The CTCF protein appears to organize the active and repressed chromatin regions into distinct architectural domains. Science, this issue p. 1017 A DNA binding protein insulates and protects repressed topological domains in the genome from adjacent active regions. Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.

The Growth Regulators warts/lats and melted Interact in a Bistable Loop to Specify Opposite Fates in Drosophila R8 Photoreceptors

Color vision in Drosophila relies on the comparison between two color-sensitive photoreceptors, R7 and R8. Two types of ommatidia in which R7 and R8 contain different rhodopsins are distributed stochastically in the retina and appear to discriminate short (p-subset) or long wavelengths (y-subset). The choice between p and y fates is made in R7, which then instructs R8 to follow the corresponding fate, thus leading to a tight coupling between rhodopsins expressed in R7 and R8. Here, we show that warts, encoding large tumor suppressor (Lats) and melted encoding a PH-domain protein, play opposite roles in defining the yR 8 or pR8 fates. By interacting antagonistically at the transcriptional level, they form a bistable loop that insures a robust commitment of R8 to a single fate, without allowing ambiguity. This represents an unexpected postmitotic role for genes controlling cell proliferation (warts and its partner hippo and salvador) and cell growth (melted).

Drosophila CRYPTOCHROME Is a Circadian Transcriptional Repressor

BACKGROUND Although most circadian clock components are conserved between Drosophila and mammals, the roles assigned to the CRYPTOCHROME (CRY) proteins are very different: Drosophila CRY functions as a circadian photoreceptor, whereas mammalian CRY proteins (mCRY1 and 2) are transcriptional repressors essential for molecular clock oscillations. RESULTS Here we demonstrate that Drosophila CRY also functions as a transcriptional repressor. We found that RNA levels of genes directly activated by the transcription factors CLOCK (CLK) and CYCLE (CYC) are derepressed in cry(b) mutant eyes. Conversely, while overexpression of CRY and PERIOD (PER) in the eye repressed CLK/CYC activity, neither PER nor CRY repressed individually. Drosophila CRY also repressed CLK/CYC activity in cell culture. Repression by CRY appears confined to peripheral clocks, since neither cry(b) mutants nor overexpression of PER and CRY together in pacemaker neurons significantly affected molecular or behavioral rhythms. Increasing CLK/CYC activity by removing two repressors, PER and CRY, led to ectopic expression of the timeless clock gene, similar to overexpression of Clk itself. CONCLUSIONS Drosophila CRY functions as a transcriptional repressor required for the oscillation of peripheral circadian clocks and for the correct specification of clock cells.

Circadian Pacemaker Neurons Transmit and Modulate Visual Information to Control a Rapid Behavioral Response

Circadian pacemaker neurons contain a molecular clock that oscillates with a period of approximately 24 hr, controlling circadian rhythms of behavior. Pacemaker neurons respond to visual system inputs for clock resetting, but, unlike other neurons, have not been reported to transmit rapid signals to their targets. Here we show that pacemaker neurons are required to mediate a rapid behavior. The Drosophila larval visual system, Bolwigs organ (BO), projects to larval pacemaker neurons to entrain their clock. BO also mediates larval photophobic behavior. We found that ablation or electrical silencing of larval pacemaker neurons abolished light avoidance. Thus, circadian pacemaker neurons receive input from BO not only to reset the clock but also to transmit rapid photophobic signals. Furthermore, as clock gene mutations also affect photophobicity, the pacemaker neurons modulate the sensitivity of larvae to light, generating a circadian rhythm in visual sensitivity.

Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis

BackgroundAmong its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites.ResultsWe tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation.ConclusionsWe have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggest that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation.

Functional Diversity of ESC-Derived Motor Neuron Subtypes Revealed through Intraspinal Transplantation

Cultured ESCs can form different classes of neurons, but whether these neurons can acquire specialized subtype features typical of neurons in vivo remains unclear. We show here that mouse ESCs can be directed to form highly specific motor neuron subtypes in the absence of added factors, through a differentiation program that relies on endogenous Wnts, FGFs, and Hh-mimicking the normal program of motor neuron subtype differentiation. Molecular markers that characterize motor neuron subtypes anticipate the functional properties of these neurons in vivo: ESC-derived motor neurons grafted isochronically into chick spinal cord settle in appropriate columnar domains and select axonal trajectories with a fidelity that matches that of their in vivo generated counterparts. ESC-derived motor neurons can therefore be programmed in a predictive manner to acquire molecular and functional properties that characterize one of the many dozens of specialized motor neuron subtypes that exist in vivo.

Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.

Iroquois complex genes induce co-expression of rhodopsins in Drosophila.

The Drosophila eye is a mosaic that results from the stochastic distribution of two ommatidial subtypes. Pale and yellow ommatidia can be distinguished by the expression of distinct rhodopsins and other pigments in their inner photoreceptors (R7 and R8), which are implicated in color vision. The pale subtype contains ultraviolet (UV)-absorbing Rh3 in R7 and blue-absorbing Rh5 in R8. The yellow subtype contains UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8. The exclusive expression of one rhodopsin per photoreceptor is a widespread phenomenon, although exceptions exist. The mechanisms leading to the exclusive expression or to co-expression of sensory receptors are currently not known. We describe a new class of ommatidia that co-express rh3 and rh4 in R7, but maintain normal exclusion between rh5 and rh6 in R8. These ommatidia, which are localized in the dorsal eye, result from the expansion of rh3 into the yellow-R7 subtype. Genes from the Iroquois Complex (Iro-C) are necessary and sufficient to induce co-expression in yR7. Iro-C genes allow photoreceptors to break the “one receptor–one neuron” rule, leading to a novel subtype of broad-spectrum UV- and green-sensitive ommatidia.