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.

miR-277 targets the proapoptotic gene-hid to ameliorate Aß42-mediated neurodegeneration in Alzheimer's model.

Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive function with no cure to date. One of the reasons for AD is the accumulation of Amyloid-beta 42 (Aß42) plaque(s) that trigger aberrant gene expression and signaling, which results in neuronal cell death by an unknown mechanism(s). Misexpression of human Aß42 in the developing retina of Drosophila exhibits AD-like neuropathology. Small non-coding RNAs, microRNAs (miRNAs), post-transcriptionally regulate the expression of their target genes and thereby regulate different signaling pathways. In a forward genetic screen, we identified miR-277 (human ortholog is hsa-miR-3660) as a genetic modifier of Aß42-mediated neurodegeneration. Loss-of-function of miR-277 enhances the Aß42-mediated neurodegeneration. Whereas gain-of-function of miR-277 in the GMR > Aß42 background downregulates cell death to maintain the number of neurons and thereby restores the retinal axonal targeting defects indicating the functional rescue. In addition, gain-of-function of miR-277 rescues the eclosion- and climbing assays defects observed in GMR > Aß42 background. Thus, gain-of-function of miR-277 rescues both structurally as well as functionally the Aß42-mediated neurodegeneration. Furthermore, we identified head involution defective (hid), an evolutionarily conserved proapoptotic gene, as one of the targets of miR-277 and validated these results using luciferase- and qPCR -assays. In the GMR > Aß42 background, the gain-of-function of miR-277 results in the reduction of hid transcript levels to one-third of its levels as compared to GMR > Aß42 background alone. Here, we provide a novel molecular mechanism where miR-277 targets and downregulates proapoptotic gene, hid, transcript levels to rescue Aß42-mediated neurodegeneration by blocking cell death. These studies shed light on molecular mechanism(s) that mediate cell death response following Aß42 accumulation seen in neurodegenerative disorders in humans and provide new therapeutic targets for neurodegeneration.

Transcriptional pausing factor M1BP regulates cellular homeostasis by suppressing autophagy and apoptosis in Drosophila eye.

During organogenesis cellular homeostasis plays a crucial role in patterning and growth. The role of promoter proximal pausing of RNA polymerase II, which regulates transcription of several developmental genes by GAGA factor or Motif 1 Binding Protein (M1BP), has not been fully understood in cellular homeostasis. Earlier, we reported that M1BP, a functional homolog of ZKSCAN3, regulates wingless and caspase-dependent cell death (apoptosis) in the Drosophila eye. Further, blocking apoptosis does not fully rescue the M1BPRNAi phenotype of reduced eye. Therefore, we looked for other possible mechanism(s). In a forward genetic screen, members of the Jun-amino-terminal-(NH2)-Kinase (JNK) pathway were identified. Downregulation of M1BP ectopically induces JNK, a pro-death pathway known to activate both apoptosis and caspase-independent (autophagy) cell death. Activation of JNK pathway components can enhance M1BPRNAi phenotype and vice-versa. Downregulation of M1BP ectopically induced JNK signaling, which leads to apoptosis and autophagy. Apoptosis and autophagy are regulated independently by their genetic circuitry. Here, we found that blocking either apoptosis or autophagy alone rescues the reduced eye phenotype of M1BP downregulation; whereas, blocking both apoptosis and autophagy together significantly rescues the M1BP reduced eye phenotype to near wild-type in nearly 85% progeny. This data suggests that the cellular homeostasis response demonstrated by two independent cell death mechanisms, apoptosis and autophagy, can be regulated by a common transcriptional pausing mechanism orchestrated by M1BP. Since these fundamental processes are conserved in higher organisms, this novel functional link between M1BP and regulation of both apoptosis and autophagy can be extrapolated to humans.

N-Acetyltransferase 9 ameliorates Aß42-mediated neurodegeneration in the Drosophila eye.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aß42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aß42 (GMR > Aß42) in developing Drosophila eye retinal neurons results in Aß42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aß42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aß42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. We found that gain-of-function of Mnat9 rescues GMR > Aß42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. Here, we propose a new neuroprotective function of Mnat9 in downregulating the JNK signaling pathway to ameliorate Aß42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aß42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9 or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aß42 and may provide new avenues for targeting neurodegeneration.

SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration.

GEMIN5 is essential for core assembly of small nuclear Ribonucleoproteins (snRNPs), the building blocks of spliceosome formation. Loss-of-function mutations in GEMIN5 lead to a neurodevelopmental syndrome among patients presenting with developmental delay, motor dysfunction, and cerebellar atrophy by perturbing SMN complex protein expression and assembly. Currently, molecular determinants of GEMIN5-mediated disease have yet to be explored. Here, we identified SMN as a genetic suppressor of GEMIN5-mediated neurodegeneration in vivo. We discovered that an increase in SMN expression by either SMN gene therapy replacement or the antisense oligonucleotide (ASO), Nusinersen, significantly upregulated the endogenous levels of GEMIN5 in mammalian cells and mutant GEMIN5-derived iPSC neurons. Further, we identified a strong functional association between the expression patterns of SMN and GEMIN5 in patient Spinal Muscular Atrophy (SMA)-derived motor neurons harboring loss-of-function mutations in the SMN gene. Interestingly, SMN binds to the C-terminus of GEMIN5 and requires the Tudor domain for GEMIN5 binding and expression regulation. Finally, we show that SMN upregulation ameliorates defective snRNP biogenesis and alternative splicing defects caused by loss of GEMIN5 in iPSC neurons and in vivo. Collectively, these studies indicate that SMN acts as a regulator of GEMIN5 expression and neuropathologies.

Protocol to study cell death using TUNEL assay in Drosophila imaginal discs.

Cell death maintains tissue homeostasis by eliminating dispensable cells. Misregulation of cell death is seen in diseases like cancer, neurodegeneration, etc. Therefore, cell death assays like TUNEL have become reliable tools, where fragmented DNA of dying cells gets fluorescently labeled and can be detected under microscope. We used TUNEL assay in Drosophila melanogaster third-instar larval eye-antennal imaginal discs to label and quantify cell death. This assay is sensitive to detect DNA fragmentation, an important event, during apoptosis in retinal neurons. For complete details on the use and execution of this profile, please refer to Wang et al. (1999), Tare et al. (2011), and Mehta et al. (2021).

Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye.

Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L 2 mutant and GMR-hid, GMR-GAL4 eye. L 2 exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L 2 and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.

Unbiased automated quantitation of ROS signals in live retinal neurons of Drosophila using Fiji/ImageJ.

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, the signal-to-noise ratio is reduced in live tissue staining. We employ the Drosophila eye model of Alzheimer's disease as a proof of concept to quantitate ROS in live tissue by adapting an unbiased method. The method presented here has a potential application for other live tissue fluorescent images.

Comparative hematopoiesis and signal transduction in model organisms.

Hematopoiesis is a continuous phenomenon involving the formation of hematopoietic stem cells (HSCs) giving rise to diverse functional blood cells. This developmental process of hematopoiesis is evolutionarily conserved, yet comparably different in various model organisms. Vertebrate HSCs give rise to all types of mature cells of both the myeloid and the lymphoid lineages sequentially colonizing in different anatomical tissues. Signal transduction in HSCs facilitates their potency and specifies branching of lineages. Understanding the hematopoietic signaling pathways is crucial to gain insights into their deregulation in several blood-related disorders. The focus of the review is on hematopoiesis corresponding to different model organisms and pivotal role of indispensable hematopoietic pathways. We summarize and discuss the fundamentals of blood formation in both invertebrate and vertebrates, examining the requirement of key signaling nexus in hematopoiesis. Knowledge obtained from such comparative studies associated with developmental dynamics of hematopoiesis is beneficial to explore the therapeutic options for hematopoietic diseases.

Hippo signaling: bridging the gap between cancer and neurodegenerative disorders.

During development, regulation of organ size requires a balance between cell proliferation, growth and cell death. Dysregulation of these fundamental processes can cause a variety of diseases. Excessive cell proliferation results in cancer whereas excessive cell death results in neurodegenerative disorders. Many signaling pathways known-to-date have a role in growth regulation. Among them, evolutionarily conserved Hippo signaling pathway is unique as it controls both cell proliferation and cell death by a variety of mechanisms during organ sculpture and development. Neurodegeneration, a complex process of progressive death of neuronal population, results in fatal disorders with no available cure to date. During normal development, cell death is required for sculpting of an organ. However, aberrant cell death in neuronal cell population can result in neurodegenerative disorders. Hippo pathway has gathered major attention for its role in growth regulation and cancer, however, other functions like its role in neurodegeneration are also emerging rapidly. This review highlights the role of Hippo signaling in cell death and neurodegenerative diseases and provide the information on the chemical inhibitors employed to block Hippo pathway. Understanding Hippo mediated cell death mechanisms will aid in development of reliable and effective therapeutic strategies in future.

Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila.

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. We found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a "no-eye" phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a "no-eye" phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. We found that upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. Our data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

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.