DevelopmentPub Date : 2024-11-25DOI: 10.1242/dev.204294
Lauren Forbes Beadle, Catherine Sutcliffe, Hilary L Ashe
{"title":"A simple MiMIC based approach for tagging endogenous genes to visualise live transcription in Drosophila.","authors":"Lauren Forbes Beadle, Catherine Sutcliffe, Hilary L Ashe","doi":"10.1242/dev.204294","DOIUrl":"https://doi.org/10.1242/dev.204294","url":null,"abstract":"<p><p>Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem loops into endogenous genes requires laborious CRISPR genome editing. Here we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging and live imaging transcription of endogenous genes and the long non-coding RNA, roX1, in the embryo. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142709333","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.203025
Raisa Bailon-Zambrano, Margaret K Keating, Emily C Sales, Abigail R Nichols, Grace E Gustafson, Colette A Hopkins, Katrinka M Kocha, Peng Huang, Lindsey Barske, James T Nichols
{"title":"The sclerotome is the source of the dorsal and anal fin skeleton and its expansion is required for median fin development.","authors":"Raisa Bailon-Zambrano, Margaret K Keating, Emily C Sales, Abigail R Nichols, Grace E Gustafson, Colette A Hopkins, Katrinka M Kocha, Peng Huang, Lindsey Barske, James T Nichols","doi":"10.1242/dev.203025","DOIUrl":"https://doi.org/10.1242/dev.203025","url":null,"abstract":"<p><p>Paired locomotion appendages are hypothesized to have redeployed the developmental program of median appendages, such as the dorsal and anal fins. Compared with paired fins, and limbs, median appendages remain surprisingly understudied. Here, we report that a dominant zebrafish mutant, smoothback(smb), fails to develop a dorsal fin. Moreover, the anal fin is reduced along the antero-posterior axis, and spine defects develop. Mechanistically, smb is caused by an insertion of a sox10:Gal4VP16transgenic construct into a non-coding region. The first step in fin, and limb, induction is aggregation of undifferentiated mesenchyme at the appendage development site. In smb, this dorsal fin mesenchyme is absent. Lineage tracing demonstrates the previously unknown developmental origin of the mesenchyme, the sclerotome, which also gives rise to the spine. Strikingly, we find that there is significantly less sclerotome in smb compared to wild type. Our results give insight into the origin and modularity of understudied median fins, which have changed position, number, size, and even disappeared across evolutionary time.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.202549
Lianna Fung, Daniel B Dranow, Arul Subramanian, Natalia Libby, Thomas F Schilling
{"title":"Cellular retinoic acid binding proteins regulate germ cell proliferation and sex determination in zebrafish.","authors":"Lianna Fung, Daniel B Dranow, Arul Subramanian, Natalia Libby, Thomas F Schilling","doi":"10.1242/dev.202549","DOIUrl":"https://doi.org/10.1242/dev.202549","url":null,"abstract":"<p><p>Cellular retinoic acid (RA) binding proteins (Crabps) solubilize intracellular RA and transport it to its nuclear receptors or cytoplasmic degradation enzymes. Despite their extreme conservation across chordates, genetic studies of Crabp function have revealed few essential functions. We have generated loss-of-function mutations in all four zebrafish Crabps and find essential roles for Crabp2s in gonad development and sex determination. Transgenic RA reporters show strong RA-responses in germ cells at the bipotential stage of gonad development. Double mutants lacking the functions of both Crabp2a and Crabp2b predominantly become male, which correlates with their smaller gonad size and reduced germ cell proliferation during gonad development at late larval and early juvenile stages. In contrast, mutants lacking the functions of both Crabp1a and Crabp1b have normal sex ratios. Exogenous RA treatments at bipotential gonad stages increase germ cell number, consistent with a direct role for RA in promoting germ cell proliferation. Our results suggest essential functions for Crabps in gonad development and sex determination.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686160","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.204256
Tim J Yeung, David G Wilkinson
{"title":"Short-range Fgf signalling patterns hindbrain progenitors to induce the neurogenesis-to-oligodendrogenesis switch.","authors":"Tim J Yeung, David G Wilkinson","doi":"10.1242/dev.204256","DOIUrl":"https://doi.org/10.1242/dev.204256","url":null,"abstract":"<p><p>In the vertebrate nervous system, neurogenesis generally precedes gliogenesis. The mechanisms driving the switch in cell type production and generation of the correct proportion of cell types remain unclear. Here, we show that Fgf20 signalling patterns progenitors to induce the switch from neurogenesis to oligodendrogenesis in the zebrafish hindbrain. Fgf20 emanating from earlier-born neurons signals at a short range to downregulate proneural gene expression in the segment centre with high spatial precision along both anterior-posterior (AP) and dorsal-ventral (DV) axes. This signal induces oligodendrocytes in the segment centre by upregulating olig2 and sox10 expression in pre-patterned competent progenitors. We show that the magnitude of proneural gene downregulation and the quantity of OPCs specified is dependent on the extent of Fgf20 signalling. Overexpression of fgf20a induces precocious specification and differentiation of oligodendrocytes among olig2+ progenitors, resulting in an increase in oligodendrocytes at the expense of neurogenesis. Thus, Fgf20 signalling defines the proportion of each cell type produced. Taken together, Fgf20 signalling from earlier-born neurons patterns hindbrain segments spatially and temporally to induce the neurogenesis-to-oligodendrogenesis switch.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.202655
Ferdinand Meneau, Pascal Lapébie, Enrico Maria Daldello, Tran Le, Sandra Chevalier, Sarah Assaf, Evelyn Houliston, Catherine Jessus, Marika Miot
{"title":"ARPP19 phosphorylation site evolution and the switch in cAMP control of oocyte maturation in vertebrates.","authors":"Ferdinand Meneau, Pascal Lapébie, Enrico Maria Daldello, Tran Le, Sandra Chevalier, Sarah Assaf, Evelyn Houliston, Catherine Jessus, Marika Miot","doi":"10.1242/dev.202655","DOIUrl":"https://doi.org/10.1242/dev.202655","url":null,"abstract":"<p><p>cAMP-PKA signaling initiates the critical process of oocyte meiotic maturation in many animals, but inhibits it in vertebrates. To address this \"cAMP paradox\", we exchanged the key PKA substrate, ARPP19, between representative species, the vertebrate Xenopus and the cnidarian Clytia, comparing its phosphorylation and function. We found that as in Xenopus, Clytia maturing oocytes undergo ARPP19 phosphorylation on a highly conserved Gwl site, which inhibits PP2A and promotes M-phase entry. In contrast, despite a PKA phosphorylation signature motif recognisable across most animals, Clytia ARPP19 was only poorly phosphorylated by PKA in vitro and in vivo. Furthermore, unlike Xenopus ARPP19, exogenous Clytia ARPP19 did not delay Xenopus oocyte maturation. We conclude that in Clytia ARPP19 does not intervene in oocyte maturation initiation because of both poor recognition by PKA and the absence of effectors that mediate vertebrate oocyte prophase arrest. We propose that ancestral ARPP19 phosphorylated by Gwl has retained a key role in M-phase across eukaryotes and it has acquired new functions during animal evolution mediated by enhanced PKA phosphorylation, allowing co-option into oocyte maturation regulation in the vertebrate lineage.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.203102
Abigail M Siniscalco, Roshan Priyarangana Perera, Jessie E Greenslade, Hemagowri Veeravenkatasubramanian, Aiden Masters, Hannah M Doll, Bushra Raj
{"title":"Barcoding Notch signaling in the developing brain.","authors":"Abigail M Siniscalco, Roshan Priyarangana Perera, Jessie E Greenslade, Hemagowri Veeravenkatasubramanian, Aiden Masters, Hannah M Doll, Bushra Raj","doi":"10.1242/dev.203102","DOIUrl":"10.1242/dev.203102","url":null,"abstract":"<p><p>Developmental signaling inputs are fundamental for shaping cell fates and behavior. However, traditional fluorescent-based signaling reporters have limitations in scalability and molecular resolution of cell types. We present SABER-seq, a CRISPR-Cas molecular recorder that stores transient developmental signaling cues as permanent mutations in cellular genomes for deconstruction at later stages via single-cell transcriptomics. We applied SABER-seq to record Notch signaling in developing zebrafish brains. SABER-seq has two components: a signaling sensor and a barcode recorder. The sensor activates Cas9 in a Notch-dependent manner with inducible control while the recorder obtains mutations in ancestral cells where Notch is active. We combine SABER-seq with an expanded juvenile brain atlas to identify cell types derived from Notch-active founders. Our data reveals rare examples where differential Notch activities in ancestral progenitors are detected in terminally differentiated neuronal subtypes. SABER-seq is a novel platform for rapid, scalable and high-resolution mapping of signaling activity during development.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686090","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.202868
Thomas M Piers, Kevin Fang, Seema C Namboori, Corin Liddle, Sally Rogers, Akshay Bhinge, Richard Killick, Steffen Scholpp
{"title":"Wnt7a-positive dendritic cytonemes control synaptogenesis in cortical neurons.","authors":"Thomas M Piers, Kevin Fang, Seema C Namboori, Corin Liddle, Sally Rogers, Akshay Bhinge, Richard Killick, Steffen Scholpp","doi":"10.1242/dev.202868","DOIUrl":"https://doi.org/10.1242/dev.202868","url":null,"abstract":"<p><p>Synaptogenesis involves the transformation of dendritic filopodial contacts into stable connections with the exact apposition of synaptic components. Signalling triggered by Wnt/β-catenin and calcium has been postulated to aid this process. However, it is unclear how such a signalling process orchestrates synapse formation to organise the spatial arrangement of synapses along dendrites. We show that Wnt7a is loaded on dynamic dendritic filopodia during spine formation in human cortical neurons. Wnt7a is present at the tips of the filopodia and the contact sites with dendrites of neighbouring neurons, triggering spatially restricted localisation of the Wnt co-receptor Lrp6. Here, we find the induction of calcium transients, the clustering of pre- and postsynaptic proteins, and the subsequent transformation into mature spines. Although soluble Wnt7a protein can also support synaptogenesis, it fails to provide this degree of spatial information for spine formation and calcium transients and synaptic markers are induced ectopically along the dendrites. Our data suggest that dendritic filopodia are Wnt7a-bearing cytonemes required for focal calcium signalling, initiate synapse formation, and provide an elegant mechanism for orchestrating the positioning of synapses along dendrites.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-22DOI: 10.1242/dev.204329
Roberta Azzarelli, Sarah Gillen, Frances Connor, Jethro Lundie-Brown, Francesca Puletti, Rosalind Drummond, Ana Raffaelli, Anna Philpott
{"title":"Phospho-regulation of ASCL1-mediated chromatin opening during cellular reprogramming.","authors":"Roberta Azzarelli, Sarah Gillen, Frances Connor, Jethro Lundie-Brown, Francesca Puletti, Rosalind Drummond, Ana Raffaelli, Anna Philpott","doi":"10.1242/dev.204329","DOIUrl":"https://doi.org/10.1242/dev.204329","url":null,"abstract":"<p><p>The proneural transcription factor ASCL1 regulates neurogenesis and drives somatic cell reprogramming into neurons. However, not all cell types can be reprogrammed by ASCL1, raising the questions of what provides competence and how we can overcome barriers to enable directed differentiation. Here, we investigate how levels of ASCL1 and its phosphorylation modulate its activity over progressive lineage restriction of embryonic stem cells. We find that inhibition of ASCL1 phosphorylation enhances reprogramming of both mesodermal and neuroectodermal cells, while pluripotent cells remain refractory to ASCL1-directed neuronal differentiation. By performing RNA-seq and ATAC-seq in neuroectoderm, we find that un(der)phosphorylated ASCL1 causes increased chromatin accessibility at sites proximal to neuronal genes, accompanied by their increased expression. Combined analysis of protein stability and proneural function of phosphomutant and phosphomimetic ASCL1 reveals that protein stability plays only a marginal role in regulating activity, while changes in amino acid charge cannot fully explain enhanced activity of the serine-proline mutant variants of ASCL1. Our work provides new insights into proneural factor activity and regulation and suggests ways to optimize reprogramming protocols in cancer and regenerative medicine.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142686161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Anterior-posterior constraint on Hedgehog signaling by hhip in teleost fin elaboration.","authors":"Yoshitaka Tanaka, Shun Okayama, Kohei Urakawa, Hidehiro Kudoh, Satoshi Ansai, Gembu Abe, Koji Tamura","doi":"10.1242/dev.202526","DOIUrl":"10.1242/dev.202526","url":null,"abstract":"<p><p>Pectoral fins, the anterior paired fins in fish, have enhanced maneuvering abilities due to morphological changes. Teleosts have fewer radial bones in their pectoral fins than basal species, resulting in more-elaborate fins. The mechanism behind this radial constraint change in teleosts is unclear. Here, we found that mutations in hhip, which encodes an antagonist of Hedgehog signaling, led to an increase in radial bones in a localized region. Expression of the Shh genes, encoding ligands of Hedgehog signaling, coincided with notable hhip expression specifically during early development. We suggest that a negative feedback effect of Hedgehog signaling by hhip regulates the constraint of the pectoral fin in zebrafish. Additionally, re-analysis of hhip-related gene expression data in zebrafish and basal species revealed that the notable hhip expression during early development is a characteristic of zebrafish that is not observed in basal species. Region-specific expression of Hox13 genes in the zebrafish pectoral fin indicated that the median region, analogous to the region with abundant radials in basal species, is expanded in hhip-/- zebrafish. These data underscore potential morphological evolution through constrained diversity.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142460555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DevelopmentPub Date : 2024-11-15Epub Date: 2024-11-26DOI: 10.1242/dev.204293
Rebecca E James, Natalie R Hamilton, Lola Nicole Huffman, Matthew P Brown, Victoria N Neckles, R Jeroen Pasterkamp, Loyal A Goff, Alex L Kolodkin
{"title":"Retinal ganglion cell-derived semaphorin 6A segregates starburst amacrine cell dendritic scaffolds to organize the mouse inner retina.","authors":"Rebecca E James, Natalie R Hamilton, Lola Nicole Huffman, Matthew P Brown, Victoria N Neckles, R Jeroen Pasterkamp, Loyal A Goff, Alex L Kolodkin","doi":"10.1242/dev.204293","DOIUrl":"10.1242/dev.204293","url":null,"abstract":"<p><p>To form functional circuits, neurons must settle in their appropriate cellular locations, and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here, we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing mouse IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate that RGCs, the first type of neuron born within the retina, play an active role in functional specialization of the IPL.</p>","PeriodicalId":11375,"journal":{"name":"Development","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142575560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}