Developmental Biology

We investigated the initial stages of head development using a new method to randomly label chicken epiblast cells with enhanced green fluorescent protein, and tracking the labeled cells. This analysis was combined with grafting mCherry-expressing quail nodes, or node-derived anterior mesendoderm (AME). These live imagings provided a new conception of the cellular mechanisms regulating brain and head ectoderm development. Virtually all anterior epiblast cells are bipotent for the development into the brain or head ectoderm. Their fate depends on the positioning after converging to the AME. When two AME tissues exist following the ectopic node graft, the epiblast cells converge to the two AME positions and develop into two brain tissues. The anterior epiblast cells bear gross regionalities that already correspond to the forebrain, midbrain, and hindbrain axial levels shortly after the node is formed. Therefore, brain portions that develop with the graft-derived AME are dependent on graft positioning.

The positioning of limbs along the anterior-posterior axis varies widely across vertebrates. The mechanisms controlling this feature remain to be fully understood. For over 30 years, it has been speculated that Hox genes play a key role in this process but evidence supporting this hypothesis has been largely indirect. In this study, we employed loss- and gain-of-function Hox gene variants in chick embryos to address this issue. Using this approach, we found that Hox4/5 genes are necessary but insufficient for forelimb formation. Within the Hox4/5 expression domain, Hox6/7 genes are sufficient for reprogramming of neck lateral plate mesoderm to form an ectopic limb bud, thereby inducing forelimb formation anterior to the normal limb field. Our findings demonstrate that the forelimb program depends on the combinatorial actions of these Hox genes. We propose that during the evolutionary emergence of the neck, Hox4/5 provide permissive cues for forelimb formation throughout the neck region, while the final position of the forelimb is determined by the instructive cues of Hox6/7 in the lateral plate mesoderm. Impact statementElucidation of the Hox code defining forelimb positioning provides novel insights in lateral plate mesoderm patterning and the integration of vertebrate column structure and limb positioning.

Annelids are regenerative animals, but the underlying mechanisms await to be discovered. Because Wnt pathway is involved in animal regeneration to varying extents, we used Aeolosoma viride to interrogate whether and how this pathway plays a role in annelid anterior regeneration. We found that the expression of wnt4, {beta}-catenin and nuclear-localized {beta}-catenin protein were up-regulated during blastemal formation and down-regulated as anterior structures gradually reformed. Consistent with potential Wnt activities in the blastema, treatments with either Wnt pathway activator (azakenpaullone) or inhibitor (XAV939) inhibited head regeneration, which further supports a role of Wnt pathway during anterior regeneration. Detailed tissue-level examines demonstrated that wound closure and blastemal cell proliferation were impaired by over-activating the pathway, and that neuronal and musculature differentiation were affected under Wnt inhibition. Combined, gene expression and chemical inhibitor data suggest the presence of dynamic Wnt activities at different anterior regeneration stages: an initial low activity may be required for wound closure, and the following activation may signal blastemal formation and cell differentiation. In a nutshell, we propose that the canonical Wnt signaling regulates blastemal cellular responses during annelid regeneration.

Hox genes establish regional identity along the anterior-posterior axis in diverse animals. Changes in Hox expression can induce striking homeotic transformations, where one region of the body is transformed into another. Previous work in Drosophila has demonstrated that Hox cross-regulatory interactions are crucial for maintaining proper Hox expression. One major mechanism is the phenomenon of "posterior prevalence", wherein anterior Hox genes are repressed by more posterior Hox genes. Loss of posterior Hox expression under this model would predict posterior-to-anterior transformations, as is frequently observed in Drosophila. While posterior prevalence is thought to occur in many animals, studies of such Hox cross-regulation have focused on a limited number of organisms. In this paper, we examine the cross-regulatory interactions of three Hox genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) in patterning thoracic and abdominal appendages in the amphipod crustacean Parhyale hawaiensis. Studies of Hox function in Parhyale have previously revealed two striking phenotypes which differed markedly from what a "posterior prevalence" model would predict, including non-contiguous and anterior-to-posterior transformations. We probe the logic of Parhyale Hox cross-regulation by using CRISPR/Cas9 to systematically examine all combinations of Ubx, abd-A, and Abd-B loss of function in Parhyale. By analyzing homeotic phenotypes and examining the expression of additional Hox genes, we reveal Hox cross-regulatory interactions in Parhyale. From these data, we also demonstrate that some Parhyale Hox genes function combinatorially to specify posterior limb identity, rather than abiding by a posterior prevalence mechanism. These results provide evidence that combinatorial Hox interactions may be responsible for the tremendous body plan diversity of crustaceans. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/485717v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@16bfc85org.highwire.dtl.DTLVardef@8f859eorg.highwire.dtl.DTLVardef@8d7892org.highwire.dtl.DTLVardef@1e6c9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neurons contribute to the complex interplay of signals that mediate heart development and homeostasis. Although a limited set of studies suggest that neuronal peptides impact vertebrate heart growth, the specific contributions of these peptides to cardiomyocyte progenitor differentiation or proliferation have not been elucidated. Here we show that the neuropeptide tachykinin along with canonical Wnt signaling regulate cardiomyocyte progenitor proliferation in the chordate model Ciona robusta. In C. robusta, the heart continues to grow throughout adulthood and classic histological studies indicate that a line of undifferentiated cells may serve as a reserve progenitor lineage. We found that this line of cardiomyocyte progenitors consists of distinct distal and midline populations. Distal progenitors divide asymmetrically to produce distal and midline daughters. Midline progenitors divide asymmetrically to produce myocardial precursors. Through single cell RNA sequencing (scRNA-seq) of adult C. robusta hearts, we delineated the cardiomyocyte progenitor expression profile. Based on this data we investigated the role of Wnt signaling in cardiomyocyte progenitor proliferation and found that canonical Wnt signaling is required to suppress excessive progenitor proliferation. The scRNA-seq data also identified a number of presumptive cardiac neural-like cells. Strikingly, we found that a subset of these neuronal cells appears to innervate the distal cardiomyocyte progenitors. Based on the expression of the tachykinin receptor in these neuronal cells, we blocked tachykinin signaling using pharmacological inhibitors and found that this drove reduced proliferation in the distal progenitor pool. Through targeted CRISPR-Cas9 knockdown we then demonstrated that both extrinsic tachykinin and intrinsic, cardiac tachykinin receptors are required for formation of the myocardial heart tube. This work provides valuable insights into how organisms may deploy neural signals to regulate organ growth in response to environmental or homeostatic inputs.

Feathers are the most complex and diverse epidermal appendages found in vertebrates. Their unique hierarchical organization and development is based on a diversity of cell types and morphologies. Despite being well characterized morphologically and extensive molecular developmental research focusing on candidate genes, little is known about the gene regulatory identities of these presumptive feather cell types. Here, we use single cell and single nuclear RNA sequencing with in situ hybridization to identify and characterize cells types in embryonic chicken feathers. We show that the distinct cell morphologies correspond to feather cell types with distinct gene expression profiles. We also describe a previously unidentified cell type, the basal barb ridge epithelium, which appears to play a role in signaling necessary for barb ridge differentiation and pulp cap production. We also analyze RNA velocity trajectories of developing feather cells, and find distinct developmental trajectories for epidermal cells that constitute the mature feather and those that function only in feather development. Finally, we produce an evolutionary tree of feather cell types based on transcription factor expression in order to test prior developmental hypotheses about feather evolution. Our tree is consistent with the developmental model of feather evolution, and sheds light on the influence of ancestral epidermal stratification on feather cell evolution. This transcriptomic approach to study feather cell types helps lay the ground work for understanding the developmental evolutionary complexity and diversity of feathers.

In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size1-3. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. One hypothesis is that the effect of hypoxia on growth and final body size is a non-adaptive consequence of the cell-autonomous effects of hypoxia on cellular metabolism. Alternatively, the effect may be an adaptive coordinated response mediated through systemic physiological mechanisms. Here we show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone, acting partially through the insulin-binding protein Imp-L2. Ecdysone is necessary to reduce growth in response to hypoxia: hypoxic growth suppression is ameliorated when ecdysone synthesis is inhibited. This hypoxia-suppression of growth is mediated by the insulin/IGF-signaling (IIS) pathway. Hypoxia reduces systemic IIS activity and the hypoxic growth-response is eliminated in larvae with suppressed IIS. Further, loss of Imp-L2, an ecdysone-response gene that suppresses systemic IIS, significantly reduces the negative effect of hypoxia on final body size. Collectively, these data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by systemic endocrine mechanisms rather than direct suppression of tissue aerobic metabolism.

Proper stem cell targeting and differentiation is necessary for regeneration to succeed. In organisms capable of whole body regeneration, considerable progress has been made identifying wound signals initiating this process, but the mechanisms that control the differentiation of progenitors into mature organs are not fully understood. Using the planarian as a model system, we identify a novel function for map3k1, a MAP3K family member possessing both kinase and ubiquitin ligase domains, to negatively regulate terminal differentiation of stem cells during eye regeneration. Inhibition of map3k1 caused the formation of multiple ectopic eyes within the head, but without controlling overall head, brain, or body patterning. By contrast, other known regulators of planarian eye patterning like WntA and notum also regulate head regionalization, suggesting map3k1 acts distinctly. Eye resection and regeneration experiments suggest that unlike Wnt signaling perturbation, map3k1 inhibition did not shift the target destination of eye formation in the animal. Instead, map3k1(RNAi) ectopic eyes emerge in the regions normally occupied by migratory eye progenitors, and the onset of ectopic eyes after map3k1 inhibition coincides with a reduction to eye progenitor numbers. Furthermore, RNAi dosing experiments indicate that progenitors closer to their normal target are relatively more sensitive to the effects of map3k1, implicating this factors in controlling the site of terminal differentiation. Eye phenotypes were also observed after inhibition of map2k4, map2k7, jnk, and p38, identifying a putative pathway through which map3k1 prevents differentiation. Together, these results suggest that map3k1 regulates a novel control point in the eye regeneration pathway which suppresses the terminal differentiation of progenitors during their migration to target destinations. Author SummaryDuring adult regeneration, progenitors must migrate and differentiate at the proper locations in order to successfully restore lost or damaged organs and tissues, yet the mechanisms underlying these abilities are not fully understood. The planarian eye is a model to study this problem, because this organ is regenerated using migratory progenitors that travel long distances through the body in an undifferentiated state prior to terminal differentiation upon their arrival at target destinations. We determined that a pathway involving the MAP kinase kinase kinase map3k1 holds planarian eye progenitors in an undifferentiated state during their transit. Inhibition of map3k1 caused a dramatic body transformation in which migratory progenitors differentiate inappropriately early, and in the wrong locations, into mature eyes. By analyzing this phenotype and measuring the change to eye progenitor abundance after map3k1 inhibition, we found that map3k1 prevents ectopic differentiation of eye cells rather than mediating body-wide patterning through the Wnt pathway. Our study argues that whole-body regeneration mechanisms involve separate steps to control patterning and progenitor differentiation.

Early neural development involves a combination of planar signals from the vertebrate organiser and vertical signals from its derived structures, the prechordal plate and notochord. However, the relative contribution of each structure to neural development is not clear. Here, we isolate anterior tissues from the primitive streak at successively later stages of development, to identify the extent of patterning that can occur prior to, during, and after the formation of the organiser and its later derivatives. Our results show that acquisition of neural identity occurs gradually and that exposure to planar signals from the developing node is necessary for neural plate specification. We also show that planar node-derived signals are required for AP patterning in isolated anterior tissues and give evidence that early neural tissue is of anterior character which subsequently becomes caudalised by signals (in part) from the developing node. However, anterior neural identity is lost without long-term contact with vertical signals from the axial mesendoderm. These results reveal a previously unappreciated level of autonomy in anterior neural development in the absence of node derived tissues. Summary statementCulture of isolated anterior tissues from the chick embryo reveal the roles of planar and vertical organiser signals for neural specification and anteroposterior patterning and maintenance.

In developing chordate embryos, the retinoic acid (RA) pathway is involved in many key developmental patterning systems. Recently, it has become clear that at least some components of the RA pathway are more ancient than chordates. However, the participation of these components in an RA pathway, and the role of such a pathway in developing non-chordate embryos remain unclear. Here, we demonstrate the presence of an extensive set of RA pathway components in the genome of the mollusc Tritia obsoleta, and examine their expression using in situ hybridization. We then examined the function of multiple RA pathway genes using RA treatments, drug treatments and morpholino (MO) knockdowns. These manipulations impacted a similar set of structures in development, especially the shell and the digestive tract, indicating that the retinoic acid pathway is functional in Tritia. Together, this is the most comprehensive evidence yet for an RA signaling pathway functioning in embryogenesis of a non-chordate.

Caenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29. ACY-2 is predominantly expressed in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose that cAMP produced by ACY-2 in the CANs acts in neighboring neurons and hypodermal cells where it activates PKA and inhibits KIN-29 to promote larval development. We discuss how this conserved pathway could be partitioned between two cells.

Craniofacial development involves the concerted action of several different tissue types and cellular processes that must be intricately regulated. Here, we describe the role of the BMP ligand Growth and Differentiation Factor 6a (gdf6a) in the development of the zebrafish craniofacial skeleton. Larval gdf6a mutant zebrafish have malformations in the midline of the craniofacial skeleton that correlate with the expression of gdf6a in the embryonic pharyngeal arches. We show that Gdf6a has arch-specific roles in craniofacial morphogenesis; Gdf6a promotes chondrogenesis and alignment of midline craniofacial elements in the posterior pharyngeal arches (arches 2 and 3) and regulates morphogenesis within the mandibular symphysis of pharyngeal arch 1. We demonstrate that Gdf6a regulates craniofacial development through activation of canonical BMP signaling, likely acting cooperatively with additional BMP ligands. Taken together, this work elucidates how Gdf6a/BMP signaling directs development of the craniofacial skeleton, specifically along the ventral midline. Summary StatementGdf6a regulates the development of the midline structures in the zebrafish craniofacial skeleton in an arch-specific manner via canonical BMP signaling.

Interneuron diversity within the central nervous system (CNS) is essential for proper circuit assembly. Functional interneurons must integrate multiple features, including combinatorial transcription factor (TF) expression, axon/dendrite morphology, and connectivity to properly specify interneuronal identity. Yet, how these different interneuron properties are coordinately regulated remains unclear. Here we used the Drosophila neural progenitor, NB5-2, known to generate late-born interneurons in a proprioceptive circuit, to determine if the early-born temporal transcription factor (TTF), Hunchback (Hb), specifies early-born interneuron identity, including molecular profile, axon/dendrite morphology, and presynapse targeting. We found that prolonged Hb expression in NB5-2 increases the number of neurons expressing early-born TFs (Nervy, Nkx6, and Dbx) at the expense of late-born TFs (Runt and Zfh2); thus, Hb is sufficient to promote interneuron molecular identity. Hb is also sufficient to transform late-born neuronal morphology to early-born neuronal morphology. Furthermore, prolonged Hb promotes the relocation of late-born neuronal presynapses to early-born neuronal presynapse neuropil locations, consistent with a change in interneuron connectivity. Finally, we found that prolonged Hb expression led to defects in proprioceptive behavior, consistent with a failure to properly specify late-born interneurons in the proprioceptive circuit. We conclude that the Hb TTF is sufficient to specify multiple aspects of early-born interneuron identity, as well as disrupt late-born proprioceptive neuron function.

Tissue development and regeneration rely on the deployment of embryonic signals to drive progenitor activity and thus generate complex cell diversity and organization. One such signal is Sonic Hedgehog (Shh), which establishes the dorsal-ventral (D/V) axis of the spinal cord during embryogenesis. However, the existence of this D/V axis and its dependence on Shh signaling during regeneration varies by species. Here we investigate the function of Shh signaling in patterning the D/V axis during spinal cord regeneration in Xenopus tropicalis tadpoles. We find that neural progenitor markers Msx1/2, Nkx6.1, and Nkx2.2 are confined to dorsal, intermediate and ventral spatial domains, respectively, in both the uninjured and regenerating spinal cord. These domains are altered by perturbation of Shh signaling. Additionally, we find that these D/V domains are more sensitive to Shh perturbation during regeneration than uninjured tissue. The renewed sensitivity of these neural progenitor cells to Shh signals represents a regeneration specific response and raises questions about how responsiveness to developmental patterning cues is regulated in mature and regenerating tissues.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish (Danio rerio), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB strain fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome or fewer than two Z chromosomes is essential to initiate oocyte development; and without the W factor or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. We use the sea urchin embryo to achieve a comprehensive understanding of miR-1s function in a developing embryo. Results indicate that miR-1 regulates gut contractions, specification, and positioning of serotonergic neurons, as well as mesodermally-derived muscles, pigment cells, and skeletogenic cells. Gain-of-function of miR-1 generally leads to more severe developmental defects than its loss-of-function. We identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Notch, Nodal, and Wnt1 signaling components. We found that miR-1s direct suppression of Nodal may indirectly regulate FoxQ2 to impact serotonergic neurons. Excess miR-1 may lead to decreased Nodal and Notch that result in decreased circumpharnygeal muscle fibers and the number of pigment cells. The striking ectopic skeletal branching induced by miR-1 mimic injections may be explained by miR-1s direct suppression of Nodal that leads to expression changes of Vegf3, and Fgfa that mediate skeletogenesis. This work demonstrates that miR-1 plays a diverse regulatory role that impacts tissues derived from all germ layers. Summary statementThis study identifies wide-ranging regulatory roles and regulatory mechanisms of miR-1 that impact structures derived from all three germ layers during embryonic development.

Limb regeneration in Ambystoma mexicanum occurs in various size fields and can recreate consistent limb morphology. The mechanism that supports such stable limb morphogenesis regardless of size is unknown. Sonic hedgehog (SHH) and fibroblasts growth factor 8 (FGF8) play important roles in anteroposterior limb patterning, similar to other tetrapods. Focusing on these two factors, we investigated the detailed expression pattern and function of Shh and Fgf8 in various blastema sizes in axolotl limb regeneration. We measured and functionally analyzed the expression domains of Shh and Fgf8 in regenerating limb blastema of various sizes, and found that, although the position and size of the Shh+ and Fgf8+ domains varied depending on the size of the blastema, the secretion of SHH was maintained at a relatively fixed working distance, regardless of blastema size. This stable secretory distance of SHH resulted in the formation of an active proliferative zone (aPZ) in the vicinity of SHH, regardless of blastema size. The aPZ was under the mitogenic influence of SHH and FGF8, resulting in high cell density in the aPZ. We also examined the impact of the aPZ on digit formation. We found that the first digit formation occurs in the aPZ. Next, the aPZ gradually shifts posteriorly as digits develop, which contributes to new digit formation at the site of the shifted aPZ. We also found that the exogenously formed aPZ caused extra digit formation even after the completion of autopod morphogenesis. Our findings suggest that the variable Shh-Fgf8 positioning in various blastema sizes causes various positioning of the aPZ, and that the aPZ leads to digit formation. The mechanism we propose here accounts for stable digit morphogenesis regardless of blastema sizes and urodele-specific digit formation. One-Sentence SummaryA unique SHH-FGF8 spatial interaction compensates for robust limb morphogenesis in various limb sizes.

BackgroundThe trigeminal ganglion (TG) is a structure of the peripheral nervous system, composed of neuronal and non-neuronal cell types, that integrates sensory input from the face and jaw. The developing TG is derived from two embryonic cell populations: neural crest and cranial placode. Both populations play critical roles in TG development and must interact to coordinate changes in gene expression that regulate specification, differentiation, and organization. However, the molecular characteristics of the heterogeneous cell populations within the developing TG remain poorly defined. ResultsWe performed single-cell RNA-sequencing (scRNA-seq) on TG from developing chick embryos at HH17. Our high-resolution dataset (14 clusters, [~]87000 cells) provides insight into cellular diversity within the developing TG. As expected, we identified placode-derived neurons as well as neural crest cells prior to neuronal differentiation. In addition to classic markers, we identified novel transcripts with unknown roles in TG development, including several long non-coding RNAs (lncRNAs). ConclusionsWe generated a single-cell atlas of the developing chick trigeminal ganglion during early axonogenesis and defined the transcriptomic states of its diverse cell populations. Our results provide a useful resource for better understanding the cell populations contributing to TG development and gene expression that drives cell identity and differentiation.

Mitochondrial activity and dynamics play crucial roles in regulating neuronal differentiation. Neural stem cells (NSCs) or neuroblasts in Drosophila require the regulation of mitochondrial fusion, along with the activity of the electron transport chain (ETC), to meet the metabolic demands of differentiation. However, the mechanisms by which mitochondrial fusion and activity together regulate NSC differentiation are not well understood. We investigated the relationship between mitochondrial fusion, and ETC complex I activity during Drosophila neuroblast differentiation. We found that depletion of complex I subunits did not affect the number of type II neuroblasts but reduced their proliferation, thereby decreasing the numbers of mature intermediate precursor cells (mINPs), ganglion mother cells (GMCs), and neurons in each lineage. Complex I depletion decreased the mitochondrial membrane potential and cristae numbers, and increased mitochondrial fragmentation and ROS. Increased ROS resulting from the depletion of antioxidant enzymes also led to a decrease in mINPs, GMCs, and neurons in each type II neuroblast lineage. Both complex I and antioxidant protein depletion led to delayed G1/S transition and decreased nuclear cyclin E levels. Interestingly, the defects in proliferation, differentiation, and ROS in complex I and anti-oxidant protein depleted neuroblasts could be restored by fused mitochondrial morphology obtained through additional depletion of the fission protein Drp1. Further, overexpression of anti-oxidant proteins could alleviate the ROS and rescue the differentiation defect in complex I depleted type II neuroblasts. Together, this study reveals a role for complex I and mitochondrial fusion in restricting ROS for differentiation in Drosophila neuroblasts. Significance statementThe electron transport chain complex I regulates differentiation in the Drosophila type II neuroblast lineage. Complex I and anti-oxidant scavenger protein depletion lead to mitochondrial fragmentation and an increase in ROS, thereby affecting the G1/S phase transition and decreasing the formation of lineage cells. Mitochondrial fusion induced by depletion of mitochondrial fission protein Drp1 in complex I and anti-oxidant protein depleted type II neuroblasts leads to suppression of the differentiation defects. This study confirms a crucial role of mitochondrial fusion in restricting ROS for appropriate division and differentiation in the type II neuroblast lineage in Drosophila.

AbstractIn binocular animals, retinal ganglion cell (RGC) axons selectively decussate at the optic chiasm. Selective decussation is directed by radial glia and midline neurons in the optic chiasm. Radial glia attach to the underlying pial basement membrane (PBM) that is rich in {beta}2 laminins. Here, we asked whether {beta}2 laminins in the PBM control the selective decussation of RGC axons. Genetic deletion of the {beta}2 subunit increased the proportion of non-decussating RGC axons in the ipsilateral tract. {beta}2 laminins are expressed in the PBM during the peak and late phases of RGC axon growth, and their deletion results in fragmentation of the PBM and dysmorphic radial glia. Consistent with the increase in the proportion of ipsilateral axons, we found persistent expression of the ipsilateral guidance cue EphrinB2 in radial glia during the late phase of axonal decussation, thus extending the developmental window for the development of the ipsilateral projection. Surprisingly, this increase in EphrinB2 was accompanied by an increase in the number of ipsilateral RGCs in the ventrotemporal retina. These results suggest that {beta}2 laminins regulate the size of the ipsilateral projection by providing cues that control the generation of ipsilateral RGCs and the expression of the ipsilateral cue EphrinB2 in the optic chiasm. Together, these findings position {beta}2 laminins as a novel regulator of the ipsilateral projection.