Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila

Journal Pre-proof Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila

Nisha, Prerna Aggarwal, Surajit Sarkar PII:

S1044-7431(19)30172-1

DOI:

https://doi.org/10.1016/j.mcn.2019.103398

Reference:

YMCNE 103398

To appear in:

Molecular and Cellular Neuroscience

Received date:

7 June 2019

Revised date:

7 August 2019

Accepted date:

25 August 2019

Please cite this article as: Nisha, P. Aggarwal and S. Sarkar, Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila, Molecular and Cellular Neuroscience (2019), https://doi.org/10.1016/j.mcn.2019.103398

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© 2019 Published by Elsevier.

Journal Pre-proof Adequate expression of Globin1 is required for development and maintenance of nervous system in Drosophila

Nisha, Prerna Aggarwal and Surajit Sarkar* Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi-110 021, India (*Correspondence: [email protected]) Abstract Neurogenesis is driven by spatially and temporally regulated proliferation of neuronal

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progenitor cells that generates enormous number of assorted neurons to drive the complex

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behavior of an organism. Drosophila nervous system provides an advantageous model for identification and elucidation of the functional significance of the novel gene(s) involved in

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neurogenesis. The present study attempts to investigate the role(s) of globin1 (glob1) in the

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development and maintenance of the nervous system in Drosophila. It is increasingly clear now that globin genes play important role(s) in the various biological phenomenon. The vertebrate

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neuroglobin has been reported to profoundly express in neuronal tissues and provides neuroprotection. We noted ubiquitous presence of Glob1 in the developing neuronal tissues with

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enhanced concentration throughout the VNC which comprises of midline cell clusters, which subsequently forms numerous types of progenitor cells and finally differentiate into specific

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neurons of the nervous system. Ubiquitous or pan-neuronal downregulation of glob1 causes partial lethality and mis-positioning of various neural-progenitor cells present in the embryonic

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midline cell clusters. Subsequently, profound expression of Glob1 was noted in the outer proliferation center of larval brain and photoreceptor axons of optic stalk. The overall arrangement of photoreceptor axons and stereotype positioning of neuroblast cells present in the central region of the brain were severally affected due to reduced expression of glob1. In addition, such larvae and surviving adults develop significant neuro-muscular disabilities. For the first time, our study suggests a novel role of glob1 in development and maintenance of the nervous system adding a new dimension to the functional significance of the multi-tasking glob1 gene in Drosophila. Keywords: Drosophila; Globin1; neurogenesis

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Journal Pre-proof 1.

Introduction The nervous system of an organism comprises great cellular diversity and form the most

complex system of the body. A developing animal needs to exploit sophisticated cellular and genetic events to ensure proper development and functioning of the nervous system. To accomplish this, it is mandatory for the cells to divide at the accurate location, time and number (Jarman, 2013; Miyares and Lee, 2018). Correspondingly, neurogenesis in Drosophila melanogaster is a well-regulated developmental process that initiates with extensive segregation events of neuronal progenitor cells from the undifferentiated ectoderm. Drosophila nervous

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system provides an advantageous model for elucidating the functional significance of novel

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gene(s) in neurogenesis, and also to investigate the cellular, molecular, physiological and behavioral aspects of neuronal and glial cells, as well as brain. Here, it is interesting to note that

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Drosophila neurons exhibit significant similarity in terms of electrophysiological and functional

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properties to the mammalian neurons (Oberst et al., 2019).

Development of the nervous system is driven by spatially and temporally regulated

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proliferation of neuronal progenitor cells that generates an enormous number of assorted neurons to drive the complex behavior of an organism. Central nervous system (CNS) development in

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Drosophila commences from a bilateral neuroectoderm which localizes on either side of a slender strip of embryonic ventral midline cells (Weiss et al., 1998). At stage 9 of

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embryogenesis, patterned delamination of neuroectodermal cells from the surface epithelium generates neural precursor cells, known as neuroblasts. Three waves of delamination generate

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three different subsets of neuroblast cell population known as SI, SII and SIII types (CamposOrtega and Harteinstein, 1957). The first wave results in formation of two rows of neuroblasts (SI) on both the sides of the median and the second wave generates SII neuroblast which fills the gap between the two rows. Third wave during stage-10 to the beginning of stage 11 of embryogenesis causes segregation of SIII neuroblasts from medial positions. These primary neuroblasts arrange themselves in four rows (1, 3, 5, and 7) and three columns (ventral, intermediate and dorsal) along the anterior-posterior (AP) and dorsoventral (DV) axis respectively, and form a structure known as Ventral Nerve Cord (VNC). Each hemisegment of VNC comprises approximately 30 neuroblasts organized in seven rows, and each neuroblast is distinguishable based on its position and molecular markers (Skeath and Thor, 2003; Technau et al., 2006; Venkatasubramanian and Mann, 2019). Expression of distinctive combination of genes 2

Journal Pre-proof in each neuroblast contributes to acquiring characteristic specification for further divisions. Drosophila VNC is a powerful model to investigate the basic mechanisms of nervous system development and function (Venkatasubramanian and Mann, 2019). The neuroblasts divide asymmetrically (in terms of cell size and fate) into one large neuroblast and a small Ganglion Mother Cell (GMC). The neuroblast retains its self-renewal capability whereas the GMCs divide only once to give rise two post-mitotic daughter cells which differentiate into neurons or glial cells (Skeath and Thor 2003; Hartenstein et al., 2008). A typical VNC is made up of ∼10,000 cells, the majority of which are neurons (Bossing et al.,

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1996; Schmidt et al., 1997; Birkholz et al., 2013). Interestingly, there is a phase of mitotic

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dormancy that commences from late embryogenesis to the end of the first instar larval stage. However, before entering to this quiescence phase, neuroblasts have been sufficiently divided to

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form the embryonic nervous system and ~10% of the future adult neurons (Prokop and Technau,

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1991; Green et al., 1993). After completion of the quiescence phase, the neuroblasts are reactivated to produce a secondary lineage of about 100 neuroblasts, which divide further and

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form the remaining 90% of adult neurons (Homem and Knoblich 2012; Kang and Reichert, 2015). Although, several genes have been identified with the important role(s) in asymmetric cell

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division, differentiation and temporal patterning of the nervous system in Drosophila; however, many aspects of nervous system development and its maintenance are still insufficiently known.

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Hemoglobins (Hbs) represent a group of evolutionarily conserved small globular proteins with the presence of typical 3-over-3 -helical sandwich structure that is also known as “globin

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fold” (Hardison, 1996). Although, the Globins have been classically regarded as a group of proteins involved in storage and circulation of gaseous oxygen; however, it is increasingly clear now that the evolutionary process has designed the globin gene family to be dynamically diverse across the animal kingdom (Vingradov and Moens, 2008; Hoffmann et al., 2010; De Henau et al., 2015). Since a long-time, only hemoglobin and myoglobin were known to represent the vertebrate globin types. Interestingly, based on the preferential expression in the nervous system, a third globin type has been established in vertebrate and named as neuroglobin (Burmester et al., 2000; Luyckx et al., 2019). Subsequently, presence of neuroglobin was observed in various mammalian, avian, reptilian, amphibian and fish species (Burmester et al., 2004). Neuroglobin is a small monomer protein with molecular weight of 16.9 kDa and exhibits characteristic feature

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Journal Pre-proof of a typical globin fold (Dewilde et al., 2001; Pesce et al., 2003). In contrast to hemoglobins and myoglobins which adopt a pentacoordinate structure and mostly expresses in red blood cells and muscles respectively, neuroglobin is a hexacoordinate protein with dominant presence in neuronal tissues (Trent and Hargrove, 2002; Burmester and Hankeln, 2009). Although, neuroglobins have been suggested to perform neuroprotective activities (Sun et al., 2001), absolute function and ancestry of neuroglobin in vertebrates is still enigmatic. Drosophila genome harbors three distinct globin genes referred to as glob1, glob2, and glob3 (Burmester and Hankeln, 1999; Burmester et al., 2006). Existence of multiple globin genes in

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Drosophila seems astonishing as it has a well-developed and adequate tracheal network to fulfill

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the oxygen demand for the small bodies and any apparent hypoxic stage is also absent during their life cycle. Therefore, it is somewhat dubious that multiple globins in Drosophila are

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exclusively involved in O2 management. In agreement to above, we have demonstrated earlier

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that adequate expression of glob1 is not only required to maintain the cellular level of Reactive Oxygen Species (ROS) but also needed to regulate several aspects of development, including

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oogenesis (Yadav et al., 2015; Yadav and Sarkar, 2018). Interestingly, during our preliminary expression studies in various larval tissues, it was noted that Glob1 accumulates profoundly in

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the Outer Proliferation Center (OPC) of the larval brain. The OPC harbors transcriptionally active cells, which give rise to different types of neuronal cells after successive divisions

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(Hofbauer and Campos-Ortega, 1990). In view of the robust presence of Glob1 in OPC, we postulated if the adequate expression of glob1 is indeed required for the development of the

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nervous system in Drosophila. Here, we demonstrate enhanced expression of Glob1 in developing neuronal tissues and also examine its role in the development and maintenance of the nervous system. 2.

Materials and Methods

2.1. Fly stocks and genetic crosses Flies were reared in standard conditions at 24○C± 1○C and 12:12 h light: dark cycle on agar/cornmeal/yeast media. Wild type strain (Oregon R)+ was used as a control for all experiments. A P-insertion line of glob1, y1w67c23;+/+;ry506P{SUPor-P}glob1KG06649/TM3,Sb (homozygous line has been conveyed by yw;+/+;P-glob1/P-glob1, in the ensuing text) is a wellestablished loss-of-function allele of glob1 (Bellen et al., 2004; Yadav et al., 2015). w;UAS4

Journal Pre-proof glob1RNAi/UAS-glob1RNAi;+/+ (Dietzl et al., 2007) and w;+/+;UAS-glob1/UAS-glob1 (Yadav et al., 2015) were used to attain RNAi mediated tissue specific downregulation or upregulation of glob1 gene respectively. w1118;+/+;Df(3R)Exel8162/TM6B,Tb is a defined deletion line spanning the entire glob1 genomic region (89A5;89A8) (Parks et al., 2004). The Elav-Gal4 (Dimitroff et al., 2012) was used to drive the UAS-transgene in pan-neuronal pattern, and insc-Gal4 in neuroblasts (Banerjee and Roy 2017). UAS-MCD8::GFP line was used to marks Gal4 driven cells in some genotypes.

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2.2. Immunohistochemistry Whole mount immunostaining in Drosophila embryos and third-instar larval tissues were

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carried out according to the protocol described earlier (Yadav et al., 2015). Primary antibodies and dilutions used for staining were rabbit anti-Drosophila Glob1 antibody (1:500) (Hankeln et

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al., 2002; Yadav et al., 2015), mouse anti-Futsch (1:100, 22C10, DSHB, USA), mouse anti-

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Fascicilin II (1:100, 1D4, DSHB, USA), mouse anti-Elav (1:100, 9F8A9, DHSB, USA), mouse anti-CNS axons (1:100, BP102, DHSB, USA), mouse anti-Chaoptin (1:100, 24B10, DHSB,

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USA) and rabbit anti-GFP (1:300, Booster, USA). Secondary antibody probes were anti-rabbit Alexa488 (1:200, Molecular Probes, USA), anti-mouse Alexa488 (1:200, Molecular Probes,

2.3. Real time PCR

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USA) and anti-mouse Cy3 (1:200, Molecular Probes, USA).

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Heads of ~100 adult flies of desired genotypes were decapitated and subjected to total RNA

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isolation using TRI reagent as per “manufactures” protocol (Cat no. T9424, Sigma-Aldrich, USA). cDNA was prepared from 2µg of total RNA using Oligo d(T)23VN (Cat no. S1327S, New England Biolabs, UK) and M-MuLV Reverse Transcriptase (Cat no. M0253S, New England Biolabs, UK). The primers used for real time PCR against glob1 and rp49 (internal control used for normalization of Ct values) were identical as described earlier in Yadav et al., 2015. Amplifications for RT PCR were carried out with 100ng of cDNA, 4µM of each primer and 1x Power SYBER Green master mix (a combination of AmpliTaq Gold DNA polymerase, deoxyribonucleotide triphosphates, and MgCl2) (BioRad, USA) in fast optical 96-well reaction plate (Applied Biosystem, USA). The thermal program comprised of an initial incubation at 98°C for 2 minutes followed by 40 cycles of 98°C for 5 seconds and 58°C for 15 seconds. The amplification reactions were run at least in triplicates on CFX96 Real-Time Detection System, 5

Journal Pre-proof Bio Rad, USA. The statistical analysis of data was applied on 2-ΔΔCt and is presented as mean ± standard deviation. 2.4. Lethality assay Lethality of different genotypes were evaluated at various developmental stages. Embryonic lethality was appraised by counting the number of brown embryos indicative of the dead ones. The number of hatched embryos were estimated after regular intervals, and subsequently, the first instars were transferred in the food vials (larval density = 10 in one food vial). Larval

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lethality was estimated by subtracting the number of pupated larvae from the total number of larvae transferred. Accordingly, the pupal lethality was calculated by recording the total number

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of adult flies eclosed and subtracting the value from the total number of pupae. Percentage

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lethality was calculated.

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2.5. Crawling assay

To observe the behavioral deficits, crawling assay was performed on third instar larvae as

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described by Nichols et al., 2012, with some modification. Late third instar larvae were collected and washed with PBS to get rid of food traces from the body surface. Further, larvae were

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transferred to 2% agar plate at a density of 2 or 3 larvae per plate. The larval movement was recorded for 1 minute using a high-speed digital camera. Captured videos were converted into

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AVI format by utilizing a MOV to AVI converter. To track the larval movement, input data was analyzed using ImageJ (NIH, USA) with a wrMTrck plugin (Dung et al., 2018). ImageJ with the

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wrMTrck plugin was also utilized to construct path track of larval movement. 2.6. Locomotor (climbing) and survival assay The climbing or Rapid Iterative Negative Geotaxis (RING) assay was performed as described earlier (Yadav et al., 2015). 1-day old flies were collected and separated into vials at maximum density of 10 flies per vial. Flies were transferred to a tube with a mark at 8 cm height, tapped and number of flies that climbed the required height in 10 seconds was recorded. The climbing ability of males and females for each genotype was recorded separately due to notable differences in their physical ability and behavior (Ali et al., 2012). The assay was performed on flies aged 1, 5, 10, 15 and 20-days and percentage of flies from each genotype that climbed 8 cm was calculated and plotted on a graph. 6

Journal Pre-proof For survival assay, adult flies of all genotypes were collected between 0 to 6 hours of eclosion and kept in fresh vials with a density of 10 flies in each. The number of flies surviving in each vial was observed and recorded on daily basis. The flies were transferred to fresh vial on an interval of every two days to avoid any death due to softness/ stickiness of food. Percentage of flies surviving after interval of every 5-days was calculated and plotted on graph. For each genotype, the survival assay was performed with 400 flies for 60 days (Bauer et al., 2004). 2.7. Microscopy and documentation

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Fluorescence stained tissues were first observed under fluorescence microscope (Olympus DP71) and subsequently imaged and analyzed using Leica TCS-SP5 II confocal microscope by

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maintaining identical parameters during comparative analysis. Similar numbers of optical sections were taken for constructing the projection images and comparative measurement of

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fluorescence intensity. Extent of co-localization between the cellular distribution pattern of two

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proteins was calculated by Leica application suite advanced fluorescence software (LAS AF Lite) (Franek et al., 2016). For this, co-localization coefficient was examined in the region of

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interest (ROI) in each genotype by selecting co-localization implement algorithm (Santos et al., 2018). Additionally, in some experiments, differential fluorescence intensity was estimated using

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ImageJ software. Total number of embryos/tissues with various cellular and staining anomalies were calculated after each staining experiment and plotted on the graph with MS Excel-2010

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2.8. Statistical analysis

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software. The images were assembled using Adobe Photoshop CS5 software.

In figures and graphs, error bars designate to the Mean ± Standard Deviation and S.E.M. (Standard Error of Mean) of all determinations. Student t-test with Bonferroni correction (α = 0.025) was performed for the analysis of the statistical difference between two groups. The differences between two groups were significant at *p-value ≤ 0.05, **p-value ≤ 0.01 and ***p≤0.001. 3.

Result

3.1. Glob1 expresses profoundly in ventral nerve cord (VNC) and peripheral nervous system during embryogenesis Cellular distribution pattern of Glob1 during embryogenesis was examined by whole-mount immunostaining. Stage-16 embryos were selected for the expression analysis since the 7

Journal Pre-proof development of embryonic nervous system is completed by then, and a fully elucidated neuronal pattern could be witnessed. During the early stages of neurogenesis, CNS midline cells arise in the embryo as two cylindrical cellular stripes and come closer at ventral midline after gastrulation at stage 5-6. These midline precursor cells go through a synchronous cell division to generate 16 midline progenitor cells per segment of VNC and differentiate to form the midline primordium (Jacobs and Goodman, 1989). At embryonic stage-16, mature midline cells are rearranged to form segmented pattern, named as thoracic neuromeres (T1-T3) and abdominal neuromeres (A1-A8) collectively known as VNC (Bossing et al., 1996). We utilized 22C10

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antibody (Canal and Ferrus, 1986) to mark the mature midline neuronal cells (Grenningloh et al.,

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1991; Seeger et al., 1993; Hummel et al., 2000).

Ubiquitous and robust presence of Glob1 was evident in the thoracic (T1-T3) and abdominal

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neuromeric (A1-A9) segments along with supportive neuroblast cells in wild type embryos (Fig.

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1A-Aʹʹ; n=350). Examination of 22C10 stained wild type embryo at lower, as well as higher magnification, revealed a stereotyped pattern of developing neuronal system where each

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neuronal cell occupies their specific position with well-patterned axonal branching (Arrowhead in Fig. 1Aʹ, also see arrow in Fig. S1Aʹ). Interestingly, an increased level of Glob1 was

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consistently evident in the midline cell clusters across VNC (arrows in Fig. 1B-Bʹʹ). This is further apparent in Fig. 1Bʹʹ which clearly displays a higher level of Glob1 in groups of 22C10

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marked midline cluster cells and estimation of co-localization coefficient suggests a perfect colocalization pattern (arrow in Fig. 1Bʹʹ, n=350; also see Fig. S1A-Aʹʹ,E,G). In addition, notable

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expression of Glob1 was perceived in the peripheral nervous system which significantly overlaps with the cells marked with 22C10 (Fig. 1A-Aʹʹ, arrow in Aʹ; also see arrows in Fig. S1A-Aʹʹ). Each segment of VNC denoted as metamere, are made up of differently mesectodermal derived cells and form a metamerically reiterated cluster (Campos-Ortega and Hartenstein, 1957). These clusters are part of the median cord which gives rise to midline glial and neuronal progenitor cells. The midline cell clusters consist of three Ventral Unpaired Medians (VUM) progenitors, two Midline Glia (MG) progenitor cells, one Median Neuroblast (MNB) and Midline Precursor 1 and 2 cells (MP1 and MP2). These midline glial progenitor cells along with VUM cells initiate the development of PNS and axonal branching after several rounds of wellregulated differentiation process (Bossing et al., 1996; Goodman et al., 1984; Jacob JR, 2000). During the subsequent examination, prominent expression of Glob1 was noted in well-organized 8

Journal Pre-proof midline cell clusters in the wild type embryos (arrows in Fig. 1B,Bʹ,Bʹʹ). Quantification of the staining intensity across different regions of VNC suggest enriched level of Glob1 in midline cells along with the cells of the lateral extremes of the VNC (Fig. S1F). On the contrary to wild type, ~97% (n=215) of P-glob1/P-glob1 embryos exhibited significant reduction in the level of Glob1 protein (compare Fig. 1A with C; also see Fig. S1D, F), which is in agreement to our earlier report that suggested over two-fold decrease in the expression of glob1 due to insertion of P-element in promoter (Yadav et al., 2015). Real time PCR further suggested ~2-fold decrease in the glob1 expression in P-glob1/P-glob1 flies (Fig.

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S1H). Since homozygous P-glob1 or P-glob1/Df(3R)Exel8162 trans-heterozygote embryos (not

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shown) displayed ubiquitous reduction in the expression of glob1, and therefore, Glob1 staining could not distinguish the 22C10 marked midline cluster cells discretely (compare Fig. 1C,D with

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Cʹ,Dʹ), and overlapping staining pattern of Glob1:22C10 was also minimal (compare Fig. S1Aʹʹ

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with Bʹʹ). Estimation of co-localization coefficient between Glob1:22C10 suggested that compared to ~35% co-localization coefficient in wild type embryos (n=398), P-glob1/P-glob1

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embryos (n=296) possess only ~21% co-localization coefficient (Fig. S1E,G). Moreover, reduced expression of glob1 caused deformed patterning of midline cell clusters (arrowhead in

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Fig. 1Cʹ and arrows in Dʹ), and dispersed positioning of midline cells across the VNC (arrows in Fig. 1Dʹ; also see arrows in Fig. S1Bʹ). It appeared that patterning defects in midline glia

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progenitor cells in neuromeric segments resulted in faulty axonal guidance and branching in mutant embryos (arrows Fig. 1Cʹ; also see arrowhead in Fig. S1Bʹ). The embryos from the

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revertant lines of P-glob1, which were generated earlier (Yadav et al., 2015) by excising the Pelement out of the genome, did not display any neuro-developmental defect. This re-confirms that associated phenotype are indeed due to P-element insertion mediated reduced expression of glob1 in the mutant embryos. Subsequently, expression level of glob1 was selectively curtailed pan neuronally by driving the UAS-glob1RNAi transgene by Elav-Gal4 and its impact on the development of nervous system was examined. Real time PCR analysis suggested ~1.6-fold decrease in the expression of glob1 in neuronal tissues (Fig. S1H). Accordingly, significant reduction in the staining intensity of Glob1 was also noted in the VNC region (circled area in Fig. 1E and also see Fig. S1C,D,F) without any substantial change in surrounding non-neuronal lateral musculature tissues (compare Fig. 1A with E). It was estimated that compared to ~35% co-localization coefficient between 9

Journal Pre-proof Glob1:22C10 staining in wild type embryos (n=398), only ~15% co-localization coefficient persisted in VNC region in UAS-glob1RNAi/Elav-Gal4 (n=395) embryos (Fig. 1Eʹʹ-Fʹʹ and also see Fig. S1Cʹʹ,E,G). Pan-neuronal depletion of glob1 caused severe defects during neuronal development in ~86% (n=194) of UAS-glob1RNAi/Elav-Gal4 embryos (Fig. 1Eʹ,Fʹ). Moreover, these embryos also displayed various anomalies such as dispersion of midline cells across the neuromeric segment and defective axonal branching (arrows in Fig. 1Eʹ,Fʹ and also see arrows and arrowheads in Fig. S1Cʹ). Intriguingly, almost a complete phenotypic rescue was achieved when the level of glob1 was reinstated by co-expressing an UAS-glob1 transgene in UAS-

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glob1RNAi/Elav-Gal4 background (Fig. S2A and F). This further suggests that tissue specific

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inadequate expression of glob1 causes defective neuronal development during embryogenesis. 3.2. Reduced expression of glob1 causes patterning defects in midline cells of CNS during

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embryogenesis

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Potential consequence(s) on the relative positioning and subsequent development of midline cells due to reduced expression of glob1 was investigated further. It has been suggested that

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especial positioning of midline cells is essential for normal development and inclusive organization of central and peripheral nervous system (Nusslein-Volhard et al., 1984; Thomas et

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al., 1988; Finkelstein et al., 1990). Interestingly, though several aspects of midline cell development are well-established, little is known about the molecules and signaling pathways

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which regulate the origin and precise positioning of these midline cells.

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In depth investigations at higher magnification suggested that localization and positioning pattern of midline precursor cells or VUM neurons were severely compromised due to both, ubiquitous or pan-neuronal downregulation of glob1 (compare Fig. 2A with B and C). Here, it is interesting to note that in-spite of their scattered positioning, the number of midline precursor cells were not affected due to the reduced level of glob1. The schematic diagram (Fig. 2H) represents the organization pattern of these precursor cells in each metamere between the ventral longitudinal tracts, which could be compared with 22C10 labeled cells in stage-16 embryo (compare Fig. 2H with G). During development VUM cells, which are derived from the midline progenitor cells localize mid-ventrally in the posterior half of each segment. The axons originating from the VUM neurons, bifurcate and extend laterally in the anterior commissures,

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Journal Pre-proof joins the intersegmental nerve (ISN) of the next anterior segment, and exit the CNS. This facilitates the formation of peripheral innervation of the neuron (Jacobs and Goodman, 1989). In depth analysis revealed gross mis-positioning and disorientation of midline cells (VUM neurons) in ~64% of P-glob1 homozygous embryos (arrowhead in Fig. 2B, also see I, n=295). The axonal projections instigating from most the embryonic segments exhibited abnormal fasciculation of the aCC neurons and localize themselves at atypical positions in ~83% (n=295) of the P-glob1/P-glob1 embryos (compare arrowhead in Fig. 2D and E). Interestingly, Elav-Gal4 driven pan-neuronal downregulation of glob1 causes more protruding defects as ~67% (n=295)

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and ~69% (n=294) of embryos displayed dispersion of midline cell clusters and misalignment of

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VUM neurons, respectively. Also, fasciculation between the axonal projections and aCC neurons and their relative positioning were severely affected in ~90% (n=294) of the embryos (compare

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arrow in Fig. 2D,E and F). It appears that dispersion of midline cells (VUM neuron) in both the

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genotypes subsequently resulted in axonal innervation towards the lateral musculature in the form of the intersegmental neuron (compare arrowhead and arrow in Fig. 2D,E and F). In

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addition to above, shortening of VNC was also evident in ~68% (n=295) and ~72% (n=294) of P-glob1 homozygous and UAS-glob1RNAi/Elav-Gal4 embryos, respectively (also see Fig. S3A-

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Cʹ). Figure 2I represents comparative graphical representation of different types of abnormalities which were observed due to ubiquitous and pan-neuronal downregulation of glob1.

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Above observations clearly demonstrate the important role of glob1 in the positioning of the

projections.

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midline cells during late stage of embryogenesis and subsequent development of axonal

3.3. Reduced expression of glob1 leads to numerous neuronal defects In order to gain further insights about the defects which originates due to reduced expression of glob1 ubiquitously, or pan-neuronally, some additional neuronal markers such as Elav, Fas-II and BP102 were used. Elav is one of the most prominent markers of the nervous system which marks almost all sets of neurons of CNS (Robinow and White, 1988). Staining with Elav in wild type embryos revealed normal development of each neuromeric segments and also divulges that each neuronal cell is well positioned and organized along the entire length of the VNC (Fig. 3AAʹʹ). During embryogenesis, developing PNS adopt a stereotyped branching pattern which could be divided in three distinct regions along the dorsal-ventral (D-V) axis: (i) dorsal cluster 11

Journal Pre-proof consisting of peripheral glia 3 (PG3), and support cells of the dorsal bipolar dendritic cells (SDBD) (ii) lateral cluster consisting of ligament cells of pentascolopidial chordotonal organ (LIG) and lateral bipolar dendritic cell (LBD) (iii) ventral cluster consisting of ventral chordotonal organ and ventral papilla (see Fig. S3G) (Halter et al., 1995; Singhania and Grueber 2014). Wild type embryos revealed the normal presence of these cluster of the cells along the DV axis (Fig. 3A-Aʹʹ). Subsequently, examination at higher magnification showed well-developed and characteristic positioning of these cells at equidistance in each neuromeric segment (arrow in Fig. 3Aʹʹ). On the contrary, ~68% (n=250) embryos from P-glob1/P-glob1 genotype exhibited

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poorly developed nervous system with distinct morphological and patterning deficits (Fig. 3B,Bʹ; also see 3J). Investigation at higher magnification revealed sparsely arranged neuronal cluster

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cells of all the above-mentioned groups, along the D-V axis (arrow in Fig. 3Bʹʹ). In addition,

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uneven distribution of NBs across the VNC was also apparent in ~70% (n=194) of the embryos (Fig. 3B-Bʹʹ). Subsequent observation in UAS-glob1RNAi/Elav-Gal4 embryos of the similar

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developmental stage revealed gross morphological defects in stereotype arrangement of various

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neuronal cells (Fig. 3C-Cʹʹ). Typical arrangement of PNS cluster cells and patterning of VNC was severely affected in ~75% of the embryos (arrow in Fig. 3Cʹʹ and also see Fig. 3J) to the

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extent that it was difficult to identify individual cell type of different neuronal clusters across the D-V axis. Here it is important to note that the abnormalities revealed by Elav staining were in

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concordance with the defects those observed by 22C10 marker (Fig. 1 and S3D,E,F). Further, by utilizing Fas-II and BP102 markers we wanted to investigate if the reduced

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expression of glob1 also influences arrangement of the longitudinal tract, embryonic midline axon guidance and PNS branching pattern (Boyle et al., 2006). Bilateral symmetry is the predominant structural organization of CNS, wherein bridging the two halves is aided by commissures (Goodman and Doe, 1993; Jacobs, 2000). The commissures are composed of interneurons which project across the midline of the VNC. In Drosophila CNS, the interneurons shoot out two distinct tracts each of which are meant for connecting the hemisegments of the CNS: the anterior commissure (AC) or the posterior commissure (PC) (Goodman and Doe, 1993; Jacobs, 2000). Each neuromere comprises of two commissures lying within the posterior half of it, except abdominal segment A9, which harbors only one commissure (Campos-Ortega and Hartenstein, 1997). Therefore, typical Drosophila VNC can be represented as a distinguishable orthogonal array of interneurons that cross the midline to join the longitudinal tracts on the other 12

Journal Pre-proof side (contralateral) and those that never cross (ipsilateral) projecting axons following a stereotypical trajectory (Garbe and Bashaw, 2004; O'Donnell et al., 2009). Wild type embryos stained with Fas-II showed the proper arrangement of the longitudinal tract (Fig. 3D) with signified properly developed longitudinal glial cells, responsible for the formation of axon projections along the VNC and longitudinal connectives (Hidalgo and Booth, 2000; Hidalgo and Brand, 1997). Ventral view of wild type embryos represents the equidistant arrangement of three ipsilateral lines on both the sides of the VNC (Fig. 3D). Often, when midline repulsion is compromised, the most medial Fas-II-positive longitudinal fascicle

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inappropriately crosses the midline and continues to extend on the opposite side (Kidd et al.,

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1998). Therefore, we investigated whether downregulation of glob1 results in the development of inappropriate Fas-II-positive bundles crossing the midline. It was observed that ubiquitous or

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pan-neuronal downregulation of glob1 did not result in any inappropriate crossovers of midline

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(Fig. 3E,F). However, P-glob1 mutant embryos showed a reduced distance between Fas-IIpositive axon fascicles of medial longitudinal connectives belonging to the opposite halves of the

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CNS, when compared with the wild type (arrowhead in Fig. 3E). Almost similar phenotype was caused when glob1 was downregulated pan-neuronally, but the distance between medial

(arrowhead in Fig. 3F).

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longitudinal connectives was minimally reduced when compared with the P-glob1 embryos

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Further, the examination of wild type embryos through lateral orientation showed the normal arrangement of axon projections (Fig. 3Dʹ) which instigated from midline cells and innervate the

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dorsal musculature (Mauss et al., 2009). These midline cells are allocated between the connectives and their axons bifurcate at the midline and exit from the ventral cord through each of the segmental nerves (SN) corresponding to VUM neurons (Goodman et al., 1984). In contrast to wild type, most lateral Fas-II-positive bundles of axons appear to stall and fail to extend laterally (arrowhead in Fig. 3Eʹ) in P-glob1 homozygous embryos. Similarly, UASglob1RNAi/Elav-Gal4 embryos displayed even more severely affected pattern of segmental neurons, in terms of disorientation of their axonal guidance and innervation (arrowhead in Fig. 3Fʹ). Distance between two medial longitudinal connectives was quantified in different genotypes and plotted on graph (Fig. 3K).

13

Journal Pre-proof We next examined CNS patterning by utilizing the BP102 neuronal marker which labels commissures (Boyle et al., 2006). In wild type embryos, the anterior and posterior commissures were observed to be well-spaced, generating a uniformly arranged reiterating pattern along the entire length of the VNC, forming a ladder-like axon scaffold (Fig. 3G). In P-glob1 homozygous embryos, improper separation of anterior and posterior commissures and rounding of spaces between them were observed (arrowhead in Fig. 3H). Interestingly, compared to P-glob1, panneuronal depletion of glob1 caused further contraction between the commissures, and therefore, distinct identification of anterior and posterior commissures became strenuous (arrowhead in Fig.

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3I). Interestingly, in-spite of above, there was no substantial change in the number of

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commissures due to global or pan-neuronal downregulation of glob1. An overall reduction in the size of the VNC in both the genotypes appears to mediate anomalous alignment of neuromeres in

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their respective embryonal segments (Fig. 3G-I). Alteration in CNS axon scaffold could be signified by additional thickening of the longitudinal connectives due to reduced expression of

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glob1 in both the genotypes (Fig. 3 compare G with H and I). In order to draw statistically

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significant information, we calculated the ratio of the distance between the longitudinal tracts in each segment of embryos to the distance between anterior and posterior commissures (Fig. 3G,

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also see L). This ratio was significantly reduced in P-glob1/P-glob1 and UAS-glob1RNAi/ElavGal4 embryos, when compared with the wild type (Table 1; Fig. 3L). Here it is also important to note that as revealed by various neuronal markers, almost a complete phenotypic rescue was

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accomplished when the level of glob1 was reinstated by co-expressing an UAS-glob1 transgene

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in UAS-glob1RNAi/Elav-Gal4 background (Fig. S2 B-E’ and G-I). Taken together, staining studies with various neuronal markers strongly suggest that an adequate level of Glob1 is required for appropriate development of the various components of the embryonic nervous system. 3.4. Glob1 expresses profoundly in larval neuronal tissues Having established the expression pattern of Glob1 and the defects that arise upon its downregulation during embryonic development; larval tissues were also evaluated under the same objective. CNS of Drosophila larvae comprises of three distinct structures: the ventral ganglion or the VNC which is known to be the functional analog of the spinal cord, the central brain that accommodates the lineages that form the mushroom bodies and two spherical optic 14

Journal Pre-proof lobes which correspond to the visual system (Lanet and Maurange, 2014). Present investigations unveiled remarkably strong signal of Glob1 in the optic stalk (OS) that connects the brain at the OPC region and the region lying below the morphogenetic furrow as revealed in both lower and higher magnification of confocal images (arrow in Fig. 4A,Aʹ) in combination with the outer OPC region of the optic lobe. The OPC region and CB harbors enormous number and diversity of neuroblast cells, similarly ommatidial cells in morphogenetic furrow exhibit active mitotic division that is cardinal for development of neuronal connections between eye and brain (Hofbaurer and Campos-Ortega, 1990; Graham et al., 2010).

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In view of deciphering the status of the structures of functional relevance, anti-Fas-II antibody

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was utilized which demarcates the neuronal network that fabricates the adult visual system (Fig. 4B,Bʹ) (Banerjee and Roy, 2018). Significant co-localization between Glob1 and Fas-II signals

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indicate the functional relevance of glob1 in the proper development of these structures

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(arrowhead in Fig. 4C,Cʹ). Similar to the embryonic stage, curtailment in abundance of glob1 at translational level was validated by staining against Glob1 protein, which was evident by the

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notably reduced staining intensity in the optic lobes and the optic stalk in larval brains procured from P-glob1/P-glob1 (arrowheads in Fig. 5A,B) and UAS-glob1RNAi/Elav-Gal4 (arrowhead in

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Fig. 5C,D). Associated morphological defects were obvious when these larval brains were costained with Fas-II. Interestingly, density and special arrangement of axons in the optic lobe were

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reduced and highly disorganized (arrowhead in Fig. 5Aʹ-Dʹ), and extent of co-localization between Glob1 and Fas-II was minimal in tissues from both the genotypes (Fig. 5Aʹʹ-Dʹʹ). This

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suggests towards the fact that downregulation of glob1 is indeed responsible for these structural manifestations. It was also noted that brains obtained from both the lines were reduced in overall size, tissue strength, and firmness as compared to those obtained from the wild type. 3.5. Reduced expression of Glob1 leads to the impaired organization of neuroblast cells In view of the fact that perturbations in glob1 expression dramatically deteriorate the structural blueprint of the VNC, it was of immense importance to evaluate how the larval brain NB progenitors are affected upon the downregulation of glob1. After the period of quiescence attained during the embryonic stage, early larval stage marks the re-entry of neuroblast into the second round of cell cycle to generate the 90% of its lineage post-embryonically (Homem and Knoblich, 2012). Neuroblasts of larval brain can be distinguished into type I, type II, mushroom 15

Journal Pre-proof body and optic lobe NBs, on the basis of their position and properties of their lineages. NBs contributing to the optic lobes evolve during the larval stages (Egger et al., 2007). NBs of the optic lobe and central brain follows a characteristic series of division to attain structural and functional maturity, illustrated by a schematic representation in Fig 6A. Type I NBs which constitute the majority of the central brain region divides in a manner comparable to NBs of the embryo. Type I NBs bud off a ganglion mother cell (GMC) and further divide to generate either two neurons or glial cells. On the other hand, Type II NBs divides asymmetrically to produce intermediate neural progenitor (INP), which possesses self-renewal potential. Mature INPs are

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produced due to a series of defined transcriptional changes and undergoes approximately three to

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five rounds of asymmetrical division post maturation, allowing them to leave more progeny neurons as compared to Type I NBs (Bello et al., 2008). Finally, the mature INPs divide to

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produce another mature INP and a GMC that eventually gives rise to either of the neurons or

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glial cells (Homem and Knoblich, 2012).

To scrutinize the consequences of reduced glob1 expression, Insc-Gal4 was exploited to

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achieve Type I and Type II NBs specific downregulation of glob1 (Banerjee and Roy, 2017). The NBs were marked with GFP by co-driving UAS-MCD8 transgene. The asymmetrical

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division is a function of differential localization of proteins and the assembly of centromere which is by the virtue of both intrinsic and extrinsic determinants. Inscuteable (Insc), an adaptor

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protein aids in attaining this polarity by interacting with another protein Bazooka which directly

2001).

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or indirectly controls spindle formation (Schaefer et al., 2000; Yu et al., 2000; Schaefer et al.,

Lethal effects, if any, due to downregulating glob1 expression under the Insc-Gal4 was investigated. Therefore, lethality assay was performed to decipher the lethal phase of UASglob1RNAi/Insc-Gal4;UAS-MCD8::GFP flies and compared with the driver control InscGal4/+;UAS-MCD8::GFP.

Continuous

examination

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UAS-glob1RNAi/Insc-Gal4;UAS-

MCD8::GFP fertilized eggs suggested 4%, 27% and 6% (n=358 ) lethality at the embryonic larval and pupal stage respectively when compared with control Insc-Gal4/+;UAS-MCD8::GFP which exhibited only 3%, 8% and 2% (n=300) lethality at equivalent stages. Since, a major proportion of UAS-glob1RNAi/Insc-Gal4;UAS-MCD8::GFP flies experienced death at the larval stage, morphological impairments in the larval brains were examined. Anti-GFP staining of 16

Journal Pre-proof larval brains of genotype UAS-glob1RNAi/Insc-Gal4;UAS-MCD8::GFP and control were performed to examine the status of type I and II NBs. Staining of brain tissue from control larvae showed a collocated arrangement of NBs and its associated cluster of clonal progenies in the OLs and a well-defined organization along the ventral ganglion (Fig. 6B-G′). Relative arrangements of these cells as a discrete cluster were clearly conveyed by counterstaining the tissues with DAPI (Fig. 6D,G). Moreover, NBs and its associated progeny cells were found to aligned properly with respect to each other (Fig. 6B,C) in control tissues. In contrast to the control, the cluster of NBs and its associated progeny cells were dispersed and their relative

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arrangement within the cluster was relatively spasmodic in UAS-glob1RNAi/Insc-Gal4;UAS-

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MCD8::GFP derived brains (Fig. 6H-M′). Compared to the regular and spherical NB cells in control tissues (arrows in Fig. 6E), downregulation of glob1 resulted in elliptical to irregular

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shape (arrows in Fig. 6K). Subsequent observation of control tissues at higher magnifications revealed typical arrangement of GMCs and other associated cluster of progeny cells aligned

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along the circumference of the central NB cell (Fig. 6Eʹ,Gʹ). In Insc-Gal4 driven downregulated

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tissue, the GMCs and other progeny cells were clustered into mass of cells occupying one side of the NB cell (Fig. 6Kʹ,Mʹ). In addition, the overall size of the brains was also reduced due to Insc-

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Gal4 mediated downregulation of glob1 (Fig. 6N). 3.6. Reduced level of glob1 disrupts the spatial arrangement of Photoreceptor axons

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Neurogenesis is followed by precise execution of axonogenesis after neural differentiation. In view of the earlier observations which suggested the pivotal role of Glob1 in the maintenance of

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the structure, number, positioning, and differentiation of NBs; we examined the status of the axonogenesis in the larval brain. Due to remarkably high expression of Glob1, we mainly focused on the axonal projections which constitute the visual network in the larval brain. In order to discern the intricacies of morphological anomalies in the multifaceted architecture of the visual system of Drosophila, anti-Chaoptin antibody was employed which specifically marks the photoreceptor cells along with the axon projections (Dung et al, 2018). The canonical structure of retina has approximately ~800 facets or ommatidia arranged in a hexagonal array. Each of these discrete ommatidia columns is composed of 8 photoreceptor cells (R-cells; R1- R8) surrounded by a sheath of pigment cells that optically isolate the unit (Waddington and Perry, 1960). The six outer rhabdomeres (R1-R6) are designated as short visual fibers whereas the inner two R7 and R8 as long visual fibers because their axonal projections innervate the laminar and 17

Journal Pre-proof deep medullary region of the optic lobe, respectively (Campos-Ortega and Strausfeld, 1972; Braitenberg and Strausfeld, 1973). The axonal projections exhibit a characteristic configuration of visual connection with the optic lobe of the brain via the optic stalk. Schematic representation of the larval brain along with eye disc, focusing on the neuronal framework (inset) that connects the photoreceptors to the optic lobes (Fig. 7E). In the wild type larvae, the geometry of the axons innervating the lamina and the medullary region formed typical ‘inverted cap’ structure (arrowhead in Fig. 7A,Aʹ). In contrast to the above, the axonal projections were defected in P-glob1/P-glob1 larvae. The characteristic

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stereotype structure was maintained to a certain extent, however, number of axons innervating

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the OPC region were found to be drastically reduced (arrowhead in Fig. 7B,Bʹ). When investigations were made in UAS-glob1RNAi/Elav-Gal4 tissues, the entire framework was

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observed to be deteriorated with the maximum anomalies in the inverted cap structure of the brain optic lobes. The structural composition of photoreceptors in the eye disc was marginally

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defected when compared to those of P-glob1/P-glob1 (compare arrowhead in Fig. 7B,Bʹ with

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C,Cʹ). It was interesting to note that besides just attaining the 3-dimensional framework of axons, the overall number of axons contributing to this sophisticated network was consistently reduced

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in the UAS-glob1RNAi/Elav-Gal4 larvae. The degree of defect in the above noted genotypes was quantified by measuring the width of the optic stalk (Fig. 7D). The bundle width in wild type

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was estimated to be ~12.4m (n=60), which was significantly reduced to a width of ~6.1m (n=75) and ~4.1m (n=80) in P-glob1/P-glob1 and UAS-glob1RNAi/Elav-Gal4 larvae,

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respectively (arrow in Fig.7A-C). Above observations evidently demonstrate that reduced expression of glob1 results in abrupt axon guidance and visual network formation. 3.7. Reduced expression of glob1 results in impaired fitness and behavioral deficits In view of several anomalies during neuronal development, manifestation of the varying expression level of glob1 was examined in aspects of perceptive and neuromuscular abilities. For above, crawling and olfaction assays in third instar larvae, and a climbing and survival assay in the adult stage were performed. The pattern observed during crawling assay suggested that compared to the majority of wild type larvae which show continuous onward movement, the larvae with reduced expression of glob1 stop suddenly, tremble their bodies and change the direction randomly. Images representing the crawling path of larvae of each genotype have been 18

Journal Pre-proof depicted in Fig 8A and plotted in graphical form in Fig 8B. Compared to 5% and 18% larvae in wild type and driver control respectively, which showed somewhat abnormal crawling pattern, 62% of homozygous P-glob1 and ~72% of UAS-glob1RNAi/Elav-Gal4 larvae exhibited significant deviation in crawling pattern (Fig. 8A,B). In addition, determination of the average speed of P-glob1/P-glob1 and UAS-glob1RNAi/Elav-Gal4 revealed a slight reduction of ~0.15mm/s and ~0.37mm/s respectively, when compared with the related controls (Supporting information S4-7). In order to determine any behavioral abnormalities in larvae due to downregulation of glob1,

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video tracking chemotaxis assay using acetone odorant was performed (Larsson et al., 2004;

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Khurana and Siddiqi 2013), and relative response index among all the genotypes was plotted in graphical form (Fig. 8C). It has been demonstrated earlier that Drosophila larva elicits attraction

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response towards chemicals of ketone family (Fishilevich et al., 2005). Compared to ~90%

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(n=50) of the wild type control larvae which exhibited distinct forward movement toward the odorant, only ~40% (n=50) of P-glob1/P-glob1 larvae could respond to the odor of acetone (Fig.

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8C), and remaining wonder aimlessly in the plate. Further, compared to ~85% (n=50) of ElavGal4/+ driver control larvae which showed explicit attraction toward acetone, only ~22% (n=50)

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larvae with pan-neuronal downregulation glob1 could retain the chemotaxis response toward odorant (Fig. 8C). Above observations suggest that adequate expression of glob1 is required to

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retain odorant sensing capabilities and chemotaxis behavior. The noteworthy defects in locomotor abilities due to reduced expression of glob1 were

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further studied by performing climbing ability test with aging. Here, it is worth noting that a previous study has already demonstrated progressive downfall of the neuromuscular abilities of P-glob1/P-glob1 adult flies (Yadav et al., 2015). Thus, adult flies with nervous system specific downregulation of glob1 were examined for locomotor activities (male and female separately) with aging and compared with Elav-Gal4/+ driver control and wild type flies of similar age, and combined average performance of male and female flies was plotted on graph (Fig. 8D). A significant decrease in neuromuscular abilities of the adult flies with pan-neuronal downregulation of glob1 was evident even on day-1 post-eclosion. Interestingly, impairments were more profoundly exhibited in male flies when compared with the adult female and this characteristic difference was persistence over aging. Subsequently, compared to the control genotypes, an enhanced rate of decline in the climbing efficiency was noted in UAS19

Journal Pre-proof glob1RNAi/Elav-Gal4 adult flies during aging (Fig. 8D). On 20-days of aging, the average climbing ability of flies reduced to 38% compared to ~58% and ~56% in wild type and driver control respectively (Fig. 8D), and such decline continued further (not shown). Above finding further establishes that glob1 plays an important role in the maintenance of neuromuscular abilities in adult flies. Further, in view of the fact that adult flies with ubiquitously reduced level of glob1 show premature aging and enhanced death (Yadav et al., 2015), we examined the effect of panneuronal downregulation of glob1 on aging and mortality, and compared with appropriate

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control flies (Fig. 8E). The adult flies from all the genotypes were upheld in identical conditions

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and in order to examine their survival rate; numbers of living flies were noted after successive interval of 5-days. Compared to wild type, post day-25 adult flies with pan-neuronal reduced

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level of glob1 started displaying striking rate of lethality (Fig. 8E). In addition, other aging

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associated impairments were frequently manifested in above flies, when compared with the control genotypes. After 50-days of aging, in comparison to ~85% (n=500) and ~80% (n=450)

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surviving adults in wild type and Elav-Gal4/+ populations respectively, only ~55% (n=550) of adult flies in UAS-glob1RNAi/Elav-Gal4 population were surviving (Fig. 8E). It is fairly evident

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from the above observations that pan-neuronal reduced expression of glob1 led to not only shortcomings in locomotor abilities but also causes enhanced aging and early death, more

Discussion

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4.

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profoundly in the male population.

It is increasingly evident now that the biological function of Globin is not restrained to only oxygen transportation and management but also intimated to perform several other biological activities. Earlier studies in various organisms have suggested that inadequate expression of glob1 causes various impairments during development including ROS imbalance, early aging, lethality, weakening of cytoskeleton integrity, and aberrant female gametogenesis (De Henau et al., 2015; Yadav et al., 2015, Yadav and Sarkar 2016, Yadav et al., 2018). In view of our earlier preliminary observation, which revealed enhanced concentration of Glob1 protein in neuronal tissues, the present study endeavors to examine the role of glob1 in neuronal development. Comprehensive expression analysis established a robust and dynamic presence of glob1 gene products in the embryonic neuronal tissues. Here, it is interesting to note that the enhanced level 20

Journal Pre-proof of Glob1 was more prevalent in the group of cells which divide further and make assorted types of cells corresponding to the larval and adult nervous system. Interestingly, global or neuronal tissue-specific reduction in the glob1 expression in P-glob1 homozygous and UASglob1RNAi/Elav-Gal4 flies respectively, exhibited more profound abnormalities in those cells in which the expression of glob1 was comparatively high. This suggests an active and important role of glob1 from seeding stages of neurogenesis. The first sign of gross irregularities due to reduced level of Glob1 appears in the VNC during stages 16 of embryogenesis, particularly in the midline cell clusters. This could again be well-

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correlated with comparatively increased level of Glob1 across the VNC which comprises midline

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cell clusters suggesting that adequate expression level of glob1 is critical for their arrangement. In this context, it is important to note that subsets of appropriately distributed midline precursor

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cells are required for initiation and maintenance of specific segmented pattern of neuromeres,

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which in turn form neuronal network during subsequent development (Schmidt et al., 1997). Intriguingly, other than midline cells, the cellular distribution pattern of Glob1 significantly

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overlaps with embryonic lateral axon branches, which constitutes the peripheral subsets of neurons, and this neuronal patterning was severely affected due to lowered level of Glob1 as

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revealed by several markers such as 22C10, Elav and Fas-II. This further suggests that Glob1 is needed to achieve accurate embryonic pattern of neuronal muscular connectivity in the periphery

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and longitudinal connectives in the CNS.

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At this stage, it is difficult to suggest a precise mechanistic role of Glob1 during neuronal development, however, in view of the fact that Glob1 has been established as an interactor of actin (Yadav et al., 2018) and actin-related protein 2/3 complex subunit 3B (Arpc3B) (Giot et al., 2003), its downregulation is likely to affect the integrity and relative arrangement of developing neuronal tissues. In this context, it is also critical to note that dynamic behavior of actin is essential for proper neurogenesis as protrusive activity of leading edge of a migrating cell during axonal pathfinding obliges regulated actin polymerization and depolymerization process (Kalil et al., 2000). Moreover, axon growth cones/shafts act as motile structure and provide sites for the formation of neuronal branches, which is characterized by rapid actin cytoskeletal reorganization (Kalil et al., 2000). In addition to above, reduced level of glob1 may also contribute to neuronal impairments by its virtue of a ROS regulator (Fordel et al., 2006; Tiedke et al., 2013; De Henau 21

Journal Pre-proof et al., 2015, Yadav et al., 2015). It has been recently demonstrated that ROS regulation is critical for various aspects of neurogenesis, i.e. formation of neuronal polarity, axonal pathfinding, the establishment of connectivity among neurons, and synaptic transmission, etc. (Oswald et al., 2018). Functioning as a signal molecule, varying cellular level of ROS modulate JNK signaling to regulate developmental apoptosis (Shen and Liu, 2006, De Henau et al., 2015). Intriguingly, GLB-12, a member of globin superfamily in C. elegans, has been demonstrated to inhibit p58/JNK MAPK pathway (De Henau et al., 2015). In this context, it is important to note that a

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developmentally-regulated neural apoptotic process is essential for maintaining the three-

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dimensional geometry of VNC as well as regulation of the degree of VNC condensation (Olofsson and Page, 2005). Enhanced apoptosis due to abrupt JNK signaling results in shortened

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and over-condensed VNC along with deformed axonal trajectories (Olofsson and Page, 2005; Ha

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et al., 2005; Qu et al., 2012; Karkali et al., 2016). These phenotypes are very much similar to those obtained due to reduced expression of glob1 in the present study. Therefore, glob1 may

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contribute in earlier stages of neurogenesis by regulating ROS mediated cellular signaling cascades, and our subsequent analysis is focused on to reveal the above aspects.

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In vertebrates, in addition to hemoglobin and myoglobin, a third form of globin, neuroglobin has been reported to profoundly express in neuronal tissues (Fabrizius et al., 2016). Interestingly,

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as noted with Glob1 in Drosophila, neuroglobin has been reported to express dynamically in mammalian brain with almost 100-fold higher expression in hypothalamus and retina, compared

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to the hippocampus and cerebral cortex (Reuss et al., 2002; Fabrizius et al., 2016). Explicit function(s) of neuroglobin in vertebrates is still uncertain, though, a few studies proposed neurodevelopmental, neuroprotective and anti-apoptotic function(s) of neuroglobin (Yu et al., 2009; Brittain 2012; Fabrizius et al., 2016; Van Acker et al., 2019). In view of the above, there is also a high possibility that glob1 might be also performing an analogous function of vertebrate neuroglobin(s) in Drosophila. Taken together, our study suggests a novel and important role of glob1 in development and maintenance of the nervous system in Drosophila. Subsequent studies aiming to unravel the specific cellular and molecular function would add a new dimension in establishing the functional significance of this multitasking gene. 22

Journal Pre-proof Acknowledgments We thank Prof. Thorsten Burmester, University of Hamburg, Germany, for Glob1 antibody, and Bloomington Stock Centre for fly stocks. Research work in the lab is supported by the grants from Council of Scientific and Industrial Research [CSIR Ref. no. 37(1667)/16/EMR-II] and Science and Engineering Research Board (SERB Ref. no. SB/EMEQ-015/2014), Government of India, New Delhi, to SS. Nisha and PA are supported by Senior and Junior research fellowship respectively, from Department of Biotechnology (DBT), Government of India, New Delhi. We also thank Delhi University for financial support under DU/DST-PURSE scheme. We are

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References

of

grateful to Ms Nabanita Sarkar for technical help.

-p

Ali YO, Ruan K, Zhai RG. NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy. Hum Mol

re

Genet. 2012, 21:237–250.

lP

Banerjee A, Roy JK. Batman regulates the axonal geometry of Drosophila larval brain by modulating actin regulator enabled. Invert Neurosci. 2018,18:1-6.

na

Banerjee A, Roy JK. Dicer-1 regulates proliferative potential of Drosophila larval neural stem cells through bantam miRNA based down-regulation of the G1/S inhibitor Dacapo. Dev Biol.

ur

2017, 423:57-65.

Jo

Bauer JH, Goupil S, Garber GB, Helfand SL. An accelerated assay for the identification of lifespan extending interventions in Drosophila melanogaster. Proc Natl Acad Sci USA. 2004, 101:12980-12985.

Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, Tsang G, et al., The BDGP gene disruption project: Single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004, 167:761-781. Bello BC, Izergina N, Caussinus E, Reichert H. Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development. Neural Dev. 2008, 3:5. Birkholz O, Rickert C, Berger C, Urbach R, Technau GM. Neuroblast pattern and identity in the Drosophila tail region and role of doublesex in the survival of sex-specific precursors. Development. 2013, 140:1830-1842. 23

Journal Pre-proof Bossing T, Udolph G, Doe CQ, Technau GM. The embryonic central nervous system lineages of Drosophila melanogaster: Dev Bio. 1996, 179:41-64. Boyle M, Nighorn A, Thomas JB. Drosophila Eph receptor guides specific axon branches of mushroom body neurons. Development. 2006, 133:1845-1854. Braitenberg V, Strausfeld NJ. Principles of the mosaic organization in the visual system's neuropil of Musca domestica. In: Jung AR (ed) Handbook of sensory physiology. Springer, Berlin Heidelberg New York, 1973, 631-659.

of

Brittain T. The Anti-Apoptotic Role of Neuroglobin. Cells. 2012, 1:1133-1155.

ro

Burmester T, Hankeln T. A globin gene of Drosophila melanogaster. Mol Biol Evol. 1999, 16:1809-1811.

-p

Burmester T, Storf J, Hasenjӓger A, Klawitter S, Hankeln T. The hemoglobin genes of

re

Drosophila. FEBS J. 2006, 273:468–480.

Burmester T, Weich B, Reinhardt S, Hankeln T. A vertebrate globin expressed in the brain.

lP

Nature. 2000, 407:520-523.

na

Campos-Ortega JA, Strausfled N. The columnar organization of the second synaptic region of the visual system of Musca domestica. Z Zellforsch. 1972, 124:561-585.

ur

Campos-Ortega JA, Hartenstein V. The embryonic development of Drosophila melanogaster. 1957, 2nd edition: xvii + 405pp.

Jo

Canal I, Ferrus A. The pattern of early neuronal differentiation in Drosophila melanogaster. J Neurogenet. 1986, 3:293-319.

De Henau S, Tilleman L, Vangheel M, Luyckx E, Trashin S, et al., A redox signalling globin is essential for reproduction in Caenorhabditis elegans. Nat Comm. 2015, 6:1-14. Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, et al., A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 2007, 448:151-156. Dimitroff B, Howe K, Watson A, Campion B, Lee HG, et al., Diet and Energy-Sensing Inputs Affect TorC1-Mediated Axon Misrouting but Not TorC2-Directed Synapse Growth in a Drosophila Model of Tuberous Sclerosis. PLoS ONE 2012, 7:e30722.

24

Journal Pre-proof Dung VM, Suong DNA, Okamaoto Y, Hiramatsu Y, Thao DTP, et al., Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting. Exp Cell Res. 2018, 366:92-102. Egger B, Boone JQ, Stevens NR, Brand AH, Doe CQ. Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe. Neural Dev. 2007, 2:1. Fabrizius A, Andre D, Laufs T, Bicker A, Reuss S, et al., Critical re-evaluation of neuroglobin

of

expression reveals conserved patterns among mammals. Neuroscience. 2016, 337:339-354. Finkelstein R, Smouse D, Capaci TM, Spradling AC, Perrimon N. The orthodenticle gene

ro

encodes a novel homeo domain protein involved in the development of the Drosophila nervous

-p

system and ocellar visual structures. Genes Dev. 1990, 4:1516-1527. Fishilevich E, Domingos AI, Asahina K, Naef F, Vosshall LB, et al., Chemotaxis behavior

re

mediated by single larval olfactory neurons in Drosophila. Curr Biol. 2005, 15:2086-2096.

lP

Fordel E, Thijs L, Martinet W, Lenjou M, Laufs T, et al., Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced

na

cell death. Neurosci Lett. 2006, 410:146-51.

Franek M, Kovaříková A, Bártová E, Kozubek S. Nucleolar Reorganization Upon Site-Specific

ur

Double-Strand Break Induction. J Histochem Cytochem. 2016, 64:669-686.

Jo

Garbe DS, Bashaw GJ. Axon guidance at the midline: from mutants to mechanisms. Crit Rev Biochem Mol Biol. 2004, 39:319-341. Giot L, Bader JS, Brouwer C, Chaudhuri A, Kuang B, et al., A protein interaction map of Drosophila melanogaster. Science. 2003, 302:1727-1736. Goodman CS, Bastiani MJ, Doe CQ, du Lac S, Helfand SL, et al., Cell recognition during neuronal development. Science. 1984, 225:1271-1279. Goodman CS, Doe CQ. Embryonic development of the Drosophila central nervous system. In the: Development of Drosophila. (ed. M. Bate and A. Martinez-Arias) Cold Spring Harbor. 1993, 2:1131-1206

25

Journal Pre-proof Graham TG, Tabei SM, Dinner AR, Rebay I. Modeling bistable cell-fate choices in the Drosophila eye: Qualitative and quantitative perspectives. Development. 2010, 137:2265–2278. Green P, Hartenstein AY, Hartenstein V. The embryonic development of the Drosophila visual system. Cell Tissue Res. 1993, 273:583-598. Ha HY, Cho IH, Lee KW, Lee KW, Song JY, et al., The axon guidance defect of the telencephalic commissures of the JSAP1-deficient brain was partially rescued by the transgenic expression of JIP1. Dev Biol. 2005: 277:184-199.

of

Halter DA, Urban J, Rickert C, Ner SS, Ito K, et al., The homeobox gene repo is required for the

melanogaster. Development. 1995, 121:317-332.

ro

differentiation and maintenance of glia function in the embryonic nervous system of Drosophila

-p

Hankeln T, Jaenicke V, Kiger L, Dewilde S, Ungerechts G, et al., Characterization of Drosophila hemoglobin. Evidence for hemoglobin-mediated respiration in insects. J Biol Chem. 2002,

re

277:29012–29017.

lP

Hardison RC. A brief history of hemoglobins: Plant, animal, protist, and bacteria. Proc Natl Acad Sci USA. 1996, 93:5675–5679.

na

Hartenstein V, Cardona A, Pereanu W, Younossi-Hartenstein A. Modeling the Developing Drosophila Brain: Rationale, Technique, and Application. BioScience. 2008, 58:823-836.

ur

Hidalgo A, Booth GE. Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS.

Jo

Development. 2000, 127:393-402.

Hidalgo A, Brand AH. Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila. Development. 1997, 124:3253-62. Hofbauer A, Campos-Ortega JA. Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster. Roux Arch Dev Biol. 1990, 198:264–274. Hoffmann FG, Opazo JC, Storz JF. Gene cooption and convergent evolution of oxygen transport hemoglobins in jawed and jawless vertebrates. Proc Natl Acad Sci USA. 2010, 107:1427414279. Homem Catarina CF, Knoblich JA. Drosophila neuroblast: a model for stem cell biology. Development. 2012, 139:4297-4310. 26

Journal Pre-proof Jacob J Roger. The Midline Glia of Drosophila: a molecular genetic model for the developmental functions of Glia. Neurobiology. 2000, 62:475-508. Jacobs JR, Goodman CS. Embryonic development of axon pathways in the Drosophila CNS. I. A glial scaffold appears before the first growth cones. J Neurosci. 1989, 9:2402-2411. Jarman A. Neurogenesis in Drosophila. In: eLS. John Wiley & Sons, Ltd: Chichester. 2013, 1-9. Kalil K, Szebenzyi G, Dent EW. Common mechanism underlying growth cone guidance and axon branching. J Neurobiol. 2000, 44:145-158.

of

Kang KH, Reichert H. Control of neural stem cell self-renewal and differentiation in Drosophila.

ro

Cell Tissue Res. 2015, 359:33-45.

Karkali K, Panayotau G, Saunders TE, Martin-Blanco E. The JNK signaling links the CNS

-p

architecture organization to motor coordination in the Drosophila embryo. bioRxiv 092486; doi:

re

https://doi.org/10.1101/092486.

Khurana S, Siddiqi O. Olfactory responses of Drosophila larvae. Chem Senses. 2013, 38:315-

lP

323.

na

Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, et al., Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance

ur

receptors. Cell. 1998; 92:205–215.

Lanet E, Maurange C. Building a brain under nutritional restriction: insights on sparing and

Jo

plasticity from Drosophila studies. Front Physiol. 2014, 5:1-10. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, Vosshall LB. Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron. 2004, 43: 703714. Luyckx E, Van Acker ZP, Ponsaerts P, Dewilde S. Neuroglobin Expression Models as a Tool to Study Its Function. Oxid Med Cell Longev. 2019, 5728129. doi: 10.1155/2019/5728129. Mauss A, Tripodi M, Evers JF, Landgraf M. Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system. PLoS Biol. 2009, 7: e1000200.

27

Journal Pre-proof Miyares RL, Lee T. Temporal control of Drosophila central nervous system development. Curr Opin Neurobiol. 2018, 56:24-32. Nichols CD, Becnel J, Pandey UB.

Methods to Assay Drosophila Behavior. Journal of

Visualized Experiments: JoVE, 2012, 61:3791-3795. Nusslein-Volhard C, Wieschaus E, Kluding H. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Wilhelm Roux Arch Dev Biol. 1984, 193:267-282.

of

Oberst P, Agirman G, Jabaudon D. Principles of progenitor temporal patterning in the

ro

developing invertebrate and vertebrate nervous system. Curr Opin Neurobiol. 2019, 56:185-193. O'Donnell M, Chance RK, Bashaw GJ. Axon Growth and Guidance: Receptor Regulation and

-p

Signal Transduction. Annu Rev Neurosci. 2009, 32:383-412.

re

Olofsson B, Page DT. Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity. Dev Biol. 2005, 279:233-243.

lP

Oswald MCW, Garnham N, Sweeney ST, Landgarf M. Regulation of neuronal development and

na

function by ROS. FEBS L. 2018, 592:679-691.

Parks AL, Cook KR, Belvin M, Dompe NA, et al., Systematic generation of high-resolution

ur

deletion coverage of the Drosophila melanogaster genome. Nat Genet. 2004, 36:288–292. Prokop A, Technau GM. The origin of postembryonic neuroblasts in the ventral nerve cord of

Jo

Drosophila melanogaster. Development. 1991, 111:79-88. Qu C, Li W, Shao Q, Dwyer Q, Huang H, et al., c-Jun N-terminal Kinase 1 (JNK1) Is Required for Coordination of Netrin Signaling in Axon Guidance. J Biol Chem. 2013, 288:1883-1895. Reuss S, Saaler-Reinhardt S, Weich B, Wystub S, Reuss MH, et al., Expression analysis of neuroglobin mRNA in rodent tissues. Neuroscience. 2002, 115:645-656. Robinow S, White K. The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev Biol. 1988, 126:294-303. Santos A, Martín P, Blasco A, Solano J, Cózar B, et al., NETs detection and quantification in paraffin embedded samples using confocal microscopy. Micron. 2018, 114:1-7.

28

Journal Pre-proof Schaefer M, Petronczki M, Dorner D, Forte M, Knoblich JA. Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system. Cell. 2001, 107:183194. Schaefer M, Shevchenko A, Shevchenko A, Knoblich JA. A protein complex containing Inscuteable and the Galpha-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr Biol. 2000, 10:353-362. Schmidt H, Rickert C, Bossing T, Vef O, Urban J, et al., The embryonic central nervous system

of

lineages of Drosophila melanogaster. II. Neuroblast lineages derived from the dorsal part of the neurectoderm. Dev Biol. 1997, 189:186-204.

ro

Sears HC, Kennedy CJ, Garrity PA. GarrityMacrophage-mediated corpse engulfment is required

-p

for normal Drosophila CNS morphogenesis. Development. 2003, 130: 3557-3565. Shen HM, Liu ZG. JNK signaling pathway is a key modulator in cell death mediated by reactive

re

oxygen and nitrogen species. Fee Radic Biol Med. 2006, 40:928-939.

lP

Singhania A, Grueber WB. Development of the embryonic and larval peripheral nervous system of Drosophila. Wiley Interdisciplinary Reviews: Dev Bio. 2014, 3:193-210.

2003, 13:8-15.

na

Skeath JB, Thor S. Genetic control of Drosophila nerve cord development. Curr Opin Neurobiol.

ur

Technau GM, Berger C, Urbach R. Generation of cell diversity and segmental pattern in the

Jo

embryonic central nervous system of Drosophila. Dev Dyn. 2006, 235:861-869. Thomas JB, Crews ST, Goodman CS. Molecular genetics of the single-minded locus: a gene involved in the development of the Drosophila nervous system. Cell. 1988, 52:133-141. Tiedke J, Cubuk C, Bermester T. Environmental acidification triggers oxidative stress and enhances globin expression in zebrafish gills. Biochem Biophys Res Comm. 2013, 441:624-629. Van Acker ZP, Luyckx E, Dewilde S. Neuroglobin expression in the brain: a story of tissue homeostasis preservation. Mol Neurobiol. 2019, 56:2101-2122. Venkatasubramanian L, Mann RS. The development and assembly of the Drosophila adult ventral nerve cord. Curr Opin Neurobiol. 2019, 56:135-143.

29

Journal Pre-proof Vinogradov SN, Moens L. Diversity of globin function: enzymatic, transport, storage, and sensing. J Biol Chem. 2008, 283:8773-8777. Waddington CH and Perry MM. The ultra-structure of the developing eye of Drosophila. Proceedings of the Royal Society of London. Series B. Biological Sciences. 1960, 153. Weiss JB, Von Ohlen T, Mellerick DM, Dressler G, Doe CQ, et al., Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity. Genes Dev. 1998, 12:3591-3602.

of

Yadav R, Kundu S, Sarkar S. Drosophila glob1 Expresses Dynamically and is Required for

ro

Development and Oxidative Stress Response. Genesis. 2015, 53:719-737. Yadav R, Nisha, Sarkar S. Drosophila globin1 is required for maintenance of the integrity of F-

-p

actin based cytoskeleton during development. Exp Cell Res. 2018, 366:16-23.

re

Yadav R, Sarkar S. Drosophila glob1 is required for the maintenance of cytoskeletal integrity during oogenesis, Dev Dyn. 2016, 245:1048–1065.

lP

Yu F, Morin X, Cai Y, Yang X, Chia W. Analysis of partner of inscuteable, a novel player of

Cell. 2000, 100:399-409.

na

Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization.

Jo

Res. 2009, 32:112-127.

ur

Yu Z, Fan X, Lo EH, Wang X. Neuroprotective roles and mechanisms of neuroglobin. Neurol

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Journal Pre-proof Figure Legends Figure 1: Enhanced expression of Glob1 (red) was evident in developing neuronal tissues. Wildtype embryo exhibiting ubiquitous expression of Glob1 with enhanced expression in VNC and embryonic musculature (A). 22C10 staining unveils the normally developed embryonic nervous system, the midline cell clusters in VNC (arrowhead) and the axonic projections (arrow) in the peripheral tissue (Aʹ), and a significant overlap was evident between staining pattern of Glob1 and 22C10 (Aʹʹ). Magnified view of VNC clearly shows enhanced level of Glob1 in VNC (B), which significantly co-localize with the normally developed midline cell clusters (arrow in B, Bʹ,

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Bʹʹ). A global reduction in the staining intensity of Glob1 was visible in P-glob1/P-glob1

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embryos (C). Ubiquitous downregulation of Glob1 in P-glob1/P-glob1 embryos with deformed neuronal cells (arrowhead and arrow in Cʹ indicate misaligned midline cluster cells and

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branching defect respectively). Drastic reduction in the degree of co-localization between Glob1

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and 22C10 signals was apparent in P-glob1/P-glob1 embryos (Cʹʹ). Magnified view of VNC also confirms reduced expression of Glob1 (D), misaligned midline cell clusters (Dʹ) and loss of

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colocalization (Dʹʹ) in P-glob1/P-glob1 embryos. Pan-neuronal downregulation of glob1 causes reduced the level of Glob1 in VNC region of UAS-glob1RNAi/Elav-Gal4 embryos (circled area

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in E, Eʹʹ), whereas somatic musculature exhibits normal expression. Impaired neuronal architecture was also evident due to VNC specific downregulation of Glob1 (Eʹ,Eʹʹ), which was

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Fʹʹ = 50µm).

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further evident under higher magnification (F,Fʹ,Fʹʹ). VNC = Ventral Nerve Cord. (Scale bar: A-

Figure 2: Magnified view of Ventral Nerve Cord displaying spatial organization and diversity of midline cells in stage-16 embryo. Wild type embryo showing the normal alignment of VUM cell with uniform spacing along the VNC (A) which was defected in P-glob1/P-glob1 (B) and UASglob1RNAi/Elav-Gal4 (C). Improper arrangement of VUM cells are indicated by arrowheads in B and C. Another optical section of the same VNC shows vMP2, dMP2, MP1 and pCC types of cells, which constitute the midline cells along with VUM cells. Arrowhead and arrow in D show the discrete arrangement of midline cells and nerve roots of segmental and inter-segmental neurons in wild type embryos, respectively. VNC of P-glob1/P-glob1 (E) and UASglob1RNAi/Elav-Gal4, (F) manifests gross deformities in comparative positioning of midline cells (arrowhead in E and F) and segmental and inter-segmental neurons (arrow in E and F). G 31

Journal Pre-proof represents a superimposed view of multiple optical sections of VNC showing different types of cells and their relative positioning in midline cell clusters. H is the schematic representation of midline cell clusters along with peripheral nerve roots. Together these cells give rise to the neurons that innervates the PNS. (I) represents a comparative estimation of different categories of defects which were scored in various genotypes. Red- Glob1; Green-22C10; VUM = Ventral Unpaired Median, pCC = Posterior Corner Cell, MP = Midline Precursor, MC = Midline Cell, AC = Anterior Commissure, PC = Posterior Commissure, con = Connectives. (Scale bar: A-G =

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10µm). Figure 3: Reduced expression of Glob1 results in numerous embryonic defects during

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neurogenesis. Elav (yellow) staining in wild type embryo represents the stereotypical arrangement of neuronal cells along the length of VNC and typical array of neuronal clusters

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along the D-V axis (A-Aʹʹ). Arrow in Aʹʹ shows the normal distribution of peripheral clusters. In

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contrast, P-glob1/P-glob1 (B-Bʹʹ) embryos exhibited deformed pattern of VNC and dispersed cell clusters of the PNS (arrow in Bʹʹ). Similarly, UAS-glob1RNAi/Elav-Gal4 (C-Cʹʹ) embryos

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represent more severally defected pattern of neuronal cells along the entire length of VNC and sparsely distributed pattern of PNS cell clusters (arrow in Cʹʹ). FAS-II (purple) staining in wild

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type embryos revealed uniformly spaced longitudinal connectives (arrowheads in D) along the midline of VNC. On the contrary, P-glob1/P-glob1 (arrowhead in E) and UAS-glob1RNAi/Elav-

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Gal4 (arrowhead in F) displayed reduced distance between the longitudinal connectives along with deformed structure compared to wild type embryos. Further, lateral view of wild type

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embryos showed normal axonal projection of segmental and intersegmental neuron (arrowhead in Dʹ) while P-glob1/P-glob1and UAS-glob1RNAi/Elav-Gal4 revealed severally defected pattern of axonal projection (arrowhead in Eʹ,Fʹ). BP102 (orange) labelled wild type embryos exhibited typical reiterating pattern, displayed as ladder-like axon scaffold (G), which is retained in both the genotypes with rounding of the spaces between the pair of ac-pc of the VNC, marked by arrowhead (H,I). (J-L) graphical representations of various defects quantifying the severity of defects generated across both downregulation lines as compared to wild type. LC = Longitudinal Connectives, SN = Segmental Nerve, ISN = Intersegmental Nerve, ac = Anterior Commissure, pc = Posterior Commissure, lo = Longitudinal connectives. (Scale bar: A,Aʹ,B,Bʹ,C,Cʹ = 50µm; Aʹʹ,Bʹʹ,Cʹʹ,D-I = 10µm).

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Journal Pre-proof Figure 4: Spatial distribution of Glob1 in third instar larval brain of wild type tissue. Larval brain along with eye imaginal disc stained for Glob1 protein (red) showed profound expression in the OPC region (arrow in A), optic stalk (arrow in Aʹ) and the region below the morphogenetic furrow. Fas-II staining (green) showed the neuronal network of the larval brain (B,Bʹ). Optic lobes and optic stalk of brain displayed substantial co-localization of Glob1 with the Fas-II positive neurons (arrowhead in C,Cʹ). OPC = Outer Proliferation Centre. (Scale bar: A,B,C = 100µm; Aʹ,Bʹ,Cʹ = 50µm).

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Figure 5: Reduced expression of Glob1 results in morphological defects in larval brain. Significant reduction in Glob1 accumulation was observed in OPC region (arrowheads in A)

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with a minimal distribution in entire brain-eye disc complex in P-glob1/P-glob1 larvae (A). Costaining with Fas-II showed the poor architecture of OPC (arrowhead in Aʹ) and the neuronal

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network (Aʹ). A reduction in Glob1:Fas-II colocalization was also evident in mutant brain

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network (Aʹʹ). Magnified view of the optic lobe and eye disc also displayed poorly developed neuronal network, and connections due to reduced level of Glob1 (B-Bʹʹ). Arrowhead in B and Bʹ

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shows reduced level of Glob1 and poorly developed optic stalk, respectively. Pan-neuronal reduced expression of Glob1 (C) caused similar defects in OPC (arrowhead C,Cʹ) in the brain

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network complex (Cʹ,Cʹʹ) and neuronal connection (D-Dʹʹ). Arrowhead in D and Dʹ points reduced the concentration of Glob1 and abridged axonal density in the optic stalk, respectively.

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OPC= Outer Proliferation Centre, OL = Optic Lobe, VG = Ventral Ganglion. (Scale bar:

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A,Aʹ,Aʹʹ,C,Cʹ,Cʹʹ = 100µm; B,Bʹ,Bʹʹ,D,Dʹ,Dʹʹ = 50µm). Figure 6: Glob1 expression is cardinal for precise neurogenesis at larval stage. Schematic diagram describing the asymmetrical mode of cell division occurring in Type I and Type II neuroblast cells (NB) in third instar larval brain (A). GFP (green) label both types of neuroblast cells which was counterstained with DAPI (red). Ventral view of whole mount larval brain of Insc-Gal4/+ showed uniformly arranged NB cells in the central brain region extending to the ventral ganglion (B-D). Magnified view of the same tissue depicted prominent GFP signal on the surface of the cell membrane, highlighting their circular shape (arrows in E). Merged projection displayed well-defined arrangement of NB cells and its associated cluster of cell progenies (G). Eʹ and Gʹ showed magnified view of a dividing NB with progeny cells. The spatial distribution of the NB cells of UAS-glob1RNAi/Insc-Gal4;UAS-MCD8::GFP 33

possessed a scattered

Journal Pre-proof configuration compared to control (H-J).

Magnified view displayed elliptical shape and

abnormal aggregation of NBs and its associated cluster of cells (arrow in K), which is also visible in the merged images (M). Kʹ and Mʹ displays magnified view of a dividing NB with irregular shape and abruptly dividing progeny cells. N is the graphical representation of the relative decrease in the size of optic lobes due to downregulation of Glob1. Red-DAPI (C,F,I,L). NB = Neuroblast, GMC = Ganglionic Mother Cell, INP = Intermediate Neural Progenitor. (Scale bar: B,C,D,H,I,J = 100µm; E,F,G,K,L,M = 50µm; Eʹ,Gʹ,Kʹ,Mʹ = 10µm).

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Figure 7: Downregulation of Glob1 induces aberrant axon targeting of photoreceptor neurons in larval brain. Chaoptin (green) staining in wild type larvae showed well-developed visual network

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(arrowhead) along with properly formed photoreceptor region below the morphogenetic furrow with a high density of axons constituting the optic stalk (arrow in A,Aʹ). On the contrary, P-

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glob1/P-glob1 larvae exhibited reduced axon density in the optic lobe (arrowhead) as well as in

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the optic stalk (arrow in B,Bʹ). Pan-neuronal depletion of glob1 resulted in more severe anomalies in inverted cap structure (arrowhead) along with reduced number of axons in the optic

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stalk (arrow in C,Cʹ). Bar graph depicts the magnitude of decrease in the bundle width across all the genotypes (D). E provides a schematic overview of third instar larval brain, highlighting the

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neuronal connections between the optic lobe and the eye imaginal disc forming an intricate 3D geometry. OL = Optic Lobe, OPC = Outer Proliferation Center, VG = Ventral Ganglion, ED =

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Eye Disc, AD = Antennal Disc, OS = Optic Stalk, M = Medullary region, L = Laminar Zone,

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PRCs = Photoreceptor Cells. (Scale bar: A,B,C = 50µm; Aʹ,Bʹ,Cʹ = 10µm). Figure 8: Modulation in Glob1 expression level manifests neuro-muscular disabilities. Crawling path of wild type and driver control displaying longer and cleaner tracks extending from the center to the periphery of the circle whereas both the mutant lines exhibited aimless and random trajectory concentrated around the center (A). The severity of crawling defect was quantified and graphically depicted as percentage of abnormal crawling paths (B). Downregulation of glob1 impairs olfaction and reception efficiency towards acetone, which was plotted as relative response index among all genotypes in graphical form (C). The histogram in D provides a comparative analysis of climbing proficiency of wild type, Elav-Gal4/+ and UASglob1RNAi/Elav-Gal4 adults. Survival assay in adult flies of wild type and Elav-Gal4/+ lines

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Journal Pre-proof showed normal survival curve, while UAS-glob1RNAi/Elav-Gal4 adults experienced early aging

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and premature death (E).

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Journal Pre-proof Table 1. Representation of quantification data of morphological defects of CNS commissure at late stage-16 and stage-17 embryos Ratio of distance between longitudinal connectives and commissures*

Genotype Wild type

2.9±0.04 (n=35) 1.83±0.02# (n=43)

w;UAS-glob1RNAi/Elav-Gal4;+/+

1.62±0.08# (n=65)

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yw;+/+,P-glob1/P-glob1

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* The distance between the longitudinal connectives and the distance between the anterior commissural bundle and posterior commissural were measured in each

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embryonic segment and the ratio of the two quantities was calculated. To avoid the

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variation of overall embryo size between the individuals during calculation, ratio of two quantities were plotted.

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# Significance compared to wild type, P<0.01 (unpaired t-test).

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n= number of segments which were measured.

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Journal Pre-proof Highlights: 

Glob1 expresses profoundly in VNC during embryogenesis.

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OPC and OS of larval brain exhibit enhanced level of Glob1.

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Depletion in the expression of glob1 causes mispositioning of various neuronal precursor cells.

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Downregulation of glob1 results in neuromuscular disabilities.

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

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