RESUMO
Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Olho/crescimento & desenvolvimento , Processamento Alternativo , Animais , Diferenciação Celular , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mutagênese Sítio-Dirigida , Células Fotorreceptoras/metabolismoRESUMO
The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Olho/crescimento & desenvolvimento , Olho/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Padronização Corporal/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genes de Insetos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Neuroepiteliais/metabolismo , Lobo Óptico de Animais não Mamíferos/crescimento & desenvolvimento , Lobo Óptico de Animais não Mamíferos/metabolismo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3⯵m diameter) alternating with thin segments (>0.1⯵m). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.
Assuntos
Drosophila melanogaster/ultraestrutura , Olho/ultraestrutura , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Células Fotorreceptoras de Invertebrados/ultraestrutura , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário , Larva/ultraestrutura , Mutação/genéticaRESUMO
Eye development requires an evolutionarily conserved group of transcription factors, termed the retinal determination network (RDN). However, little is known about the molecular mechanism by which the RDN instructs cells to differentiate into photoreceptors. We show that photoreceptor cell identity in Drosophila is critically regulated by the transcription factor Glass, which is primarily expressed in photoreceptors and whose role in this process was previously unknown. Glass is both required and sufficient for the expression of phototransduction proteins. Our results demonstrate that the RDN member Sine oculis directly activates glass expression, and that Glass activates the expression of the transcription factors Hazy and Otd. We identified hazy as a direct target of Glass. Induced expression of Hazy in the retina partially rescues the glass mutant phenotype. Together, our results provide a transcriptional link between eye field specification and photoreceptor differentiation in Drosophila, placing Glass at a central position in this developmental process.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Neurogênese/fisiologia , Células Fotorreceptoras de Invertebrados/citologia , Animais , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Retina/citologia , Retina/embriologiaRESUMO
Recent studies have revealed that hippocampal area CA2 plays an important role in hippocampal network function. Disruption of this region has been implicated in neuropsychiatric disorders. It is well appreciated that cholinergic input to the hippocampus plays an important role in learning and memory. While the effect of elevated cholinergic tone has been well studied in areas CA1 and CA3, it remains unclear how changes in cholinergic tone impact synaptic transmission and the intrinsic properties of neurons in area CA2. In this study, we applied the cholinergic agonist carbachol and performed on-cell, whole-cell, and extracellular recordings in area CA2. We observed that under conditions of high cholinergic tone, CA2 pyramidal neurons depolarized and rhythmically fired bursts of action potentials. This depolarization depended on the activation of M1 and M3 cholinergic receptors. Furthermore, we examined how the intrinsic properties and action-potential firing were altered in CA2 pyramidal neurons treated with 10 µM carbachol. While this intrinsic burst firing persisted in the absence of synaptic transmission, bursts were shaped by synaptic inputs in the intact network. We found that both excitatory and inhibitory synaptic transmission were reduced upon carbachol treatment. Finally, we examined the contribution of different channels to the cholinergic-induced changes in neuronal properties. We found that a conductance from Kv7 channels partially contributed to carbachol-induced changes in resting membrane potential and membrane resistance. We also found that D-type potassium currents contributed to controlling several properties of the bursts, including firing rate and burst kinetics. Furthermore, we determined that T-type calcium channels and small conductance calcium-activated potassium channels play a role in regulating bursting activity.
Assuntos
Potenciais de Ação/fisiologia , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Muscarínicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio Tipo T/metabolismo , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Estimulação Elétrica/métodos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiologia , Camundongos , Células Piramidais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Across metazoans, visual systems employ different types of photoreceptor neurons (PRs) to detect light. These include rhabdomeric PRs, which exist in distantly related phyla and possess an evolutionarily conserved phototransduction cascade. While the development of rhabdomeric PRs has been thoroughly studied in the fruit fly Drosophila melanogaster, we still know very little about how they form in other species. To investigate this question, we tested whether the transcription factor Glass, which is crucial for instructing rhabdomeric PR formation in Drosophila, may play a similar role in other metazoans. Glass homologues exist throughout the animal kingdom, indicating that this protein evolved prior to the metazoan radiation. Interestingly, our work indicates that glass is not expressed in rhabdomeric photoreceptors in the planarian Schmidtea mediterranea nor in the annelid Platynereis dumerilii. Combined with a comparative analysis of the Glass DNA-binding domain, our data suggest that the fate of rhabdomeric PRs is controlled by Glass-dependent and Glass-independent mechanisms in different animal clades.
RESUMO
Development of the insect compound eye requires a highly controlled interplay between transcription factors. However, the genetic mechanisms that link early eye field specification to photoreceptor terminal differentiation and fate maintenance remain largely unknown. Here, we decipher the function of 2 transcription factors, Glass and Hazy, which play a central role during photoreceptor development. The regulatory interactions between Glass and Hazy suggest that they function together in a coherent feed-forward loop in all types of Drosophila photoreceptors. While the glass mutant eye lacks the expression of virtually all photoreceptor genes, young hazy mutants correctly express most phototransduction genes. Interestingly, the expression of these genes is drastically reduced in old hazy mutants. This age-dependent loss of the phototransduction cascade correlates with a loss of phototaxis in old hazy mutant flies. We conclude that Glass can either directly or indirectly initiate the expression of most phototransduction proteins in a Hazy-independent manner, and that Hazy is mainly required for the maintenance of functional photoreceptors in adult flies.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Olho Composto de Artrópodes/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
The ability of some animals to regrow their head and brain after decapitation provides a striking example of the regenerative capacity within the animal kingdom. The acoel worm Symsagittifera roscoffensis can regrow its head, brain and sensory head organs within only a few weeks after decapitation. How rapidly and to what degree it also reacquires its functionality to control behavior however remains unknown. We provide here a neuroanatomical map of the brain neuropils of the adult S. roscoffensis and show that after decapitation a normal neuroanatomical organization of the brain is restored in the majority of animals. By testing different behaviors we further show that functionality of both sensory perception and the underlying brain architecture are restored within weeks after decapitation. Interestingly not all behaviors are restored at the same speed and to the same extent. While we find that phototaxis recovered rapidly, geotaxis is not restored within 7â weeks. Our findings show that regeneration of the head, sensory organs and brain result in the restoration of directed navigation behavior, suggesting a tight coordination in the regeneration of certain sensory organs with that of their underlying neural circuits. Thus, at least in S. roscoffensis, the regenerative capacity of different sensory modalities follows distinct paths.