Molecular Genetics of Axial Patterning, Growth and Disease in the Drosophila Eye

University, Indore, India Ph. D Drosophila Genetics , D. The fruit fly, Drosophila melanogaster , eye serves as an excellent model to study cell type specification during development. Drosophila eye has been extensively used to address diverse biological processes like patterning cell proliferation, cell death, cell survival, polarity and genetic basis of human diseases.

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The compound eye of an adult fly develops from the primordium called eye imaginal disc harbored inside larva, which initiates from a group of cells as early as an embryo. Axial patterning is hallmark of organogenesis which results in transition of a single sheet of cells into a 3-dimensional organ. Our lab is interested in understanding the molecular genetic basis of the Dorso-ventral patterning, the first lineage restriction event of early eye primordium.

DV patterning thus plays a crucial role in inducing growth and patterning of early eye disc. The dorsal and ventral domains of the eye are generated by the domain specific expression and function of the dorsal selector genes and the ventral growth controlling genes. Our lab will focus on identifying new components of DV patterning and their role in retinal determination of the eye.

Our laboratory seek to provide a better understanding of the molecular, genetic, and environmental basis of normal eye development, as well as elucidate the genes and molecules that when altered result in the genesis of birth defects in eye. AD manifests as a gradual decline of cognitive functions of learning and memory due to selective atrophy of specific cell populations in central and peripheral nervous system. One of the causes of cytotoxicity seen in AD is generation and accumulation of amyloid-beta plaques in the brain.

Several animal models have been developed to understand the molecular genetic, chemical basis of this disease.

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September 30, ISBN Mechanistic insights into generation of axes in the developing eye. Epub Nov The above findings did not rule out, however, that other ato cis -elements might drive an Ato-dependent phase of expression in the BO primordium. Nonetheless, our extensive analysis of the ato regulatory DNA could not identify any other fragment sufficient to drive Ato-dependent expression in the BOP Figs. In addition, the CR3 region did not harbor an Ato-dependent element because knock-out of the proneural phase of ato expression by mutation of the So2 site did not uncover any residual enhancer function Fig.

Taken together, these results rendered the maintenance of ato through autoregulation unlikely.

To scrutinize the apparent autoregulation-independent maintenance of ato expression in the primary precursors of the BO, we decided to directly test whether this phase of endogenous ato expression required the prior activity of Ato at the proneural stage. Hence, we assessed expression of endogenous ato at the mRNA and protein levels in both wt and ato 1 mutant embryos.

By analogy with the eye disc, we predicted the loss of the second phase of ato expression but not the early proneural phase, in ato 1 mutant embryos Jarman et al. Our results were similar to the previously described dynamic pattern of ato expression Daniel et al. Surprisingly, however, we found that the expression of ato did not change in the ato 1 mutant BOP Fig. Not only was the proneural phase of gene expression unchanged Fig.

Once again, the results were consistent between mRNA and protein. These results conclusively show that autoregulation is not a preeminent feature of ato expression in the BOP, and stand in contrast to ato regulation in the CE and OC primordia.

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A—B and E—F in situ hybridization with ato -specific probe. In all panels the BOP or primary neuronal precursors are marked by a white dashed outline or a yellow arrow. Expression of the ato mRNA is detected first in a broad domain A and then becomes restricted to a few cells B. This raised the possibility that the md fragment containing CR3 controlled both phases of ato transcription, initiation and maintenance. B Summary diagram of the enhancers controlling ato expression in the visual organs of Drosophila.

C cis -regulation of ato in relation to the phylogenetic context. In conclusion, the Ato-dependent maintenance phase of ato expression documented in the developing CE and OC does not occur in the larval visual organ primordium. In the BOP, the extended expression of ato in the primary neurons is not autoregulatory and the core ato BO enhancer remains the sole cis -regulatory element identified that is both required and sufficient to induce expression in the developing BO. The sum of our findings defines the core control region ato BO for ato expression in the developing larval eye of Drosophila Fig.

Four salient features of ato BO are to be noted: I it maps outside the cis -regulatory regions shared by CE and OC; II it is located upstream of the ato transcription unit; III it is directly controlled by the visual system specification factor So at two sites; IV consistent with a surprising lack of ato autoregulation in the BOP, it does not include an autoregulatory component. In the Drosophila larval eye, primordium specification and ato expression depend on the RD factor So and its partner Eya Cheyette et al.

As we show here, and in agreement with genetic data, transcriptional activation of ato in the BO involves direct binding of So at the ato BO enhancer Fig. Two sites, So1 and So2, mediate this regulation Fig. Loss of the So1 site lead to a partial reduction in reporter expression Fig. A possible molecular mechanism for these findings is that binding of the So transcription factor to the So2 site promotes recruitment at the So1 site, and not vice versa. Thus, loss of So2 would result in loss of all So protein recruitment at the enhancer, whereas loss of So1 would not.

S4 and thus is likely to bind other important regulators that may contribute to the unequal recruitment of the So protein to its cis -binding sites. Whereas our findings are consistent with the developmental genetics of BO formation, a surprising result is that the ato BO enhancer is not part of the shared control regions for onset of ato expression in the CE and OC.

Another striking finding of our study is the lack of ato autoregulation. However surprising, multiple lines of evidence show this to be the case. Second, no other region of the ato genomic DNA displays ato -dependent activity see summaries in Figs. Altogether this evidence demonstrates the genuine lack of an ato feedback loop in the BO.

Autoregulation of proneural gene expression is not a feature shared by all proneural factors, e. Similarly, among vertebrate proneural homologues, direct auto-regulation is present in Math1 but not in Mash1 or Neurogenin Helms et al. Thus, the lack of auto-regulation of a pronerual factor is far from unprecedented.

In the BO, this lack of autoregulation may reflect the less precise constitution of the BO, with its smaller and variable number of photoreceptors. In contrast, the exquisite precision of the ommatidial array and embryonic CHOs or the prolonged and repetitive recruitment of precursors in the large femoral CHO may have benefited from the establishment of robust positive auto-regulatory loops. Since Ato controls the development of a diverse set of sensory organs, it may utilize both modes of dynamic gene regulation.

Lastly, another, long noted, distinct feature of the BO concerns its independence of Pax6 input Suzuki and Saigo, Embryos lacking both ey and toy , however, form apparently normal larval eyes Suzuki and Saigo, Thus, ato BO does not require a Pax6 input for gene activation.

In summary, the ato BO enhancer presents multiple distinctive features that raise a number of interesting questions as to its evolutionary trajectory. Comparative evidence assigns different evolutionary ages to the three serially homologous visual organs of Drosophila Fig. The origin of OC and CE from a singleton precursor visual organ predates the diversification of arthropods begun at least Mya. The BO thus represents the most recent of the three sense organs of the Drosophila visual system and is more closely related to the CE than to the OC Fig.

This phylogenetic framework is useful for inferring the likely origin of the BO-specific enhancer ato BO. The most parsimonious explanation for use of the same genomic regulatory regions of ato in CE and OC Fig. By extension, the newly discovered ato BO CRM is evolutionarily more recent and hence of novel origin. Its sequence conservation between D. The reduction of larval head morphology in higher Diptera Cyclorrhapha , a likely causative influence in the reorganization of the BO CRM see below , suggests a maximal age of million years Wiegmann et al. Within this phylogenetic framework, the use of direct transcriptional activation by So emerges as an ancestral and evolutionarily conserved feature of the ato BO cis -element; whereas, the dispensability of Pax6 in BO development and ato expression represents an outcome of regressive evolution at the gene regulatory level Porter and Crandall, ; Friedrich, and Both conclusions have intriguing implications.

Mechanistically, at least two possibilities appear conceivable. The ato BO cis -element may have originated by tandem duplication of the preexisting regulatory elements for ato induction in the CE and OC, followed by disuse of the Pax6 and other unnecessary sites in the new copy. Alternatively, the ato BO originated de novo. The latter could have occurred in two ways, either via cooption of preexisting CRMs or shadow enhancers, or via utilization of novel nascent CRMs.

Although challenging, comparative promoter studies in a larger sample of dipteran species could yield insights into which of the above trajectories is more likely. The evolutionarily derived regression of Pax6 input, by contrast, was likely caused by the reduction of the larval head in the more recent evolutionary past of higher Diptera. Independent insights into the latter aspect have been gained through comparative studies of head and visual sense organ development in the red flour beetle Tribolium castaneum.

Like Drosophila , this species develops indirectly and forms simple larval eyes, homologous to the BOs, during embryogenesis. However, unlike in Drosophila , development of the Tribolium larval eye requires the same ancestral combination of RD factors including Ey, Toy, Eya, and So as the compound eyes of the adult Yang et al.

In addition, these same factors are also involved in the development of the Tribolium larval head Luan et al.

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This is in contrast to the reduced and internalized head of the Drosophila larva, which is a hallmark trait of higher Diptera. Taking all in consideration, it was concluded that in higher dipteran species, like Drosophila , the profound reduction of the larval head and the associated modification of the genetic network for head development relinquished the requirement for Pax6 in the ocular head segment, which also gives rise to the BO Luan et al.

Indeed, the reduction of Pax6 expression in the developing larva might even have necessitated the evolution of a Pax6-independent ato control element for the BO. By a similar comparative logic, the absence of autoregulation represents another regressive aspect of the evolution of ato regulation. Autoregulation is an ancestral feature of ato control since it is utilized by ato not only in CE and OC but also in the chordotonal stretch receptors Sun et al. As for the Pax6 input, reduction of the larval head of Drosophila and structural regression of the BO to a simpler morphology most likely led to the disuse and eventual loss of the ancestral autoregulatory mechanism.

Interestingly, based on the suspected link between head and BO reduction in higher dipteran flies, we previously predicted that RDN target gene regulation would be profoundly modified in the BO and prioritized the ato gene as testing ground for this idea Luan et al. While the comparative evidence explains how developmental body-plan evolution facilitated or necessitated novel regulation of ato in the BOP, it remains to be understood how the ato BO CRM originated at the sequence level.

It is thus exciting to note that the testability of both Pax6 and Ato input regression in other species identifies ato as a novel paradigm for studying the evolution of cis -regulatory logic in highly diversified paralogous organs Carroll et al. Cook for comments on the manuscript. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.

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The publisher's final edited version of this article is available at Dev Biol. See other articles in PMC that cite the published article. Associated Data Supplementary Materials 1. Introduction Higher Diptera, like Drosophila , utilize three sense organs to navigate the visual environment: Open in a separate window. Role and regulation of ato in the Drosophila larval eye In all figures, embryos or larvae are shown with anterior to the left and dorsal up; yellow arrows point to BO. Materials and methods Fly lines and transgenics For the list of lines used see Fig.

Histology In all experiments, multiple independent transgenic lines were analyzed for each construct, as reported in Fig. Results Analysis of candidate cis-regulatory regions for ato transcription To begin our search for ato BO enhancers, we first explored two genomic regions that were previously reported to confer ato gene expression in the BO marked ato-RE and 3. The core region for BO expression maps to a bp evolutionarily conserved sequence A Diagram of genomic regions tested for enhancer activity and summary of results in wt and ato 1 mutant embryos.

The ato-RE cis-element is not an autoregulatory enhancer of the BO Since ato expression in the primary neuronal precursors is maintained through an autoregulatory loop in the CE and OC primordia, we next investigated whether the ato-RE enhancer Fig. Lack of autoregulatory phase of ato expression in the BOP The above findings did not rule out, however, that other ato cis -elements might drive an Ato-dependent phase of expression in the BO primordium.

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Discussion The sum of our findings defines the core control region ato BO for ato expression in the developing larval eye of Drosophila Fig. Distinctive features of the ato BO enhancer In the Drosophila larval eye, primordium specification and ato expression depend on the RD factor So and its partner Eya Cheyette et al. Cis -sites for retinal determination factor Sine oculis mediate onset of ato in BO. Supplementary Material 1 Click here to view. Acknowledgments We thank D. Evolution of proneural atonal expression during distinct regulatory phases in the developing Drosophila eye.

Regulation of ocellar specification and size by twin of eyeless and homothorax. From DNA to diversity: The spitz gene is required for photoreceptor determination in the Drosophila eye where it interacts with the EGF receptor.

Drosophila as a developmental paradigm of regressive brain evolution: A Compound Eye Relict. Singh A, Kango-Singh M, editors. Genetic regulation of early eye development in non-dipteran insects. The head involution defective gene of Drosophila melanogaster functions in programmed cell death. Autoregulation and multiple enhancers control Math1 expression in the developing nervous system. Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos. Atonal is the Proneural Gene for Drosophila Photoreceptors. The Pax6 genes eyeless and twin of eyeless are required for global patterning of the ocular segment in the Tribolium embryo.