For post-docs pursuing an academic career, the transition to a group leader position can be a notoriously difficult one, not least because the availability of necessary support and training for this varies so greatly between labs and institutions. In an effort to provide an alternative support structure for postdoctoral researchers entering the academic job market, and help build a network of young group leaders in the developmental biology field, Development launched its Pathway to Independence (PI) programme in 2022. Eight PI fellows are chosen annually, not just on the strength of their scientific record and vision, but also on how much they would benefit from the programme's peer-support networks, leadership training, mentorship opportunities and profile-raising features in Development. In this Perspective, I discuss the impact the PI programme has for its fellows, with a particular focus on the inaugural 2023 cohort, as well as the research community more widely.

The origin of complete metamorphosis in insects is one of the major unresolved mysteries of insect evolution. The proposed juvenile stage regulator, chronologically inappropriate morphogenesis (chinmo), may provide some insights into the evolution of metamorphosis. Here, we examined the function of Chinmo in the hemimetabolous milkweed bug, Oncopeltus fasciatus. chinmo and br were co-expressed throughout the nymphal stage in this species. chinmo knockdown in these insects resulted in enhanced rate of wing pad growth and cuticular morphogenesis, and the appearance of characteristics seen in older nymphal instars, ultimately leading to precocious adult development through the upregulation of Ecdysone-induced protein 93F. The enhanced wing pad growth of chinmo knockdown nymphs could be rescued through the knockdown of br, although br expression was not altered when chinmo was knocked down. We propose that during the evolution of holometaboly, the expression and functions of chinmo and br became temporally separated to create the unique larval specific and pupal morphologies. Furthermore, our findings demonstrate that nymphal stages can be compressed into fewer instars, supporting the notion that insect metamorphosis evolved through drastic heterochronic shifts in life history stages.

The narrowest biological tubes are comprised of cells that hollow to form an intracellular lumen. Here, we examine early lumenogenesis of the C. elegans excretory cell, which branches to form an H-shaped intracellular tube spanning the length of the worm. Using genetically paralyzed embryos to freeze movement, we describe lumen initiation and branching for the first time using time-lapse fluorescence microscopy. We show that the excretory cell lumen forms through a plasma membrane invasion mechanism when a nascent lumen grows from the plasma membrane into the cytoplasm. The lumen subsequently extends along the left-right axis before branching to form anterior-posterior projections. Through a genetic screen, we identify mutations in ina-1/⍺-integrin and pat-3/β-integrin that block lumenogenesis at the anterior-posterior branching step, and we show that integrin function is required within the excretory cell. Finally, we find that the excretory cell crosses the epidermal basement membrane where anterior-posterior branches form and demonstrate that basement membrane crossing fails in integrin mutant embryos. Our findings reveal how an intracellular lumen initiates and branches and identify integrins and basement membrane as key branching regulators.

Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable organ size control model. Gain-of-function of voltage-gated K+ channels in fibroblast-lineage blastema cells or inhibition of the Ca2+-dependent phosphatase calcineurin causes dramatic fin regenerative overgrowth. However, Ca2+ fluxes and their potential origins from dynamic membrane voltages have not been directly linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating fins to identify widespread Ca2+ transients in distal blastema cells. Membrane depolarization of isolated fin fibroblasts triggered voltage-gated Ca2+ channel-dependent Ca2+ spikes. Single cell transcriptomics identified cacna1c (L-type channel), cacna1ba (N-type) and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca2+ signaling. Small molecule inhibition revealed that L- and/or N-type channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. GCaMP imaging showed Cacna1g enables Ca2+ dynamics in the distal blastema. We conclude that 'bioelectricity' for fin size control reflects voltage-modulated Ca²+ fluxes in fibroblast-lineage blastemal cells that decelerate outgrowth at a rate tuned to restore the original scale of the fin.

Neural crest induction begins early during neural plate formation, requiring precise transcriptional control to activate lineage-specific enhancers. Here, we demonstrate that SALL4, a transcription factor associated with syndromes featuring craniofacial anomalies, plays a critical role in early cranial neural crest (CNCC) specification. Using SALL4-het-KO human iPSCs to model clinical haploinsufficiency, we show that SALL4 directly recruits BAF to CNCC-lineage specific enhancers at the neuroectodermal stage, specifically when neural crest gene expression is induced at the neural plate border. Without functional SALL4, BAF is not loaded at chromatin, leaving CNCC enhancers inaccessible. Consequently, the cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression, despite normal neuroectodermal and neural plate progression. Moreover, by performing SALL4 isoform-specific depletion, we demonstrate that the SALL4A is the isoform essential for CNCC induction and specification, and that SALL4B cannot compensate for SALL4A loss in this developmental process. In summary, our findings reveal SALL4 as essential regulator of BAF-dependent enhancer activation during early stages of neural crest development, providing molecular insights into SALL4-associated craniofacial anomalies.

Adherens junctions (AJs) stabilize cell contacts by coupling adhesion molecules to the cytoskeleton. AJ proteins have been studied extensively in epithelia, but less is known about their roles in other cell types. Here, we describe a role for AJ proteins in C. elegans glia. Previously, we showed that C. elegans glia use the adhesion molecule SAX-7/L1CAM to anchor the dendritic endings of URX and BAG sensory neurons at the nose during embryo elongation, allowing their dendrites to stretch to their full lengths. Using cell-specific rescue and depletion experiments, we show that the AJ proteins MAGI-1 and HMR-1/Cadherin also act in glia to promote URX and BAG dendrite extension. MAGI-1 is a multi-PDZ domain protein that can simultaneously interact with PDZ-binding (PB) motifs in SAX-7 and HMP-2/β-catenin, thus potentially bridging SAX-7 to the cadherin-catenin complex. The SAX-7 PB motif also binds AFD-1/Afadin. Double mutant analyses indicate that many of these players act redundantly, consistent with parallel interactions among them. As MAGI-1, HMR-1, and AFD-1 are all found in epithelial AJs, we propose that an AJ-like complex in glia promotes dendrite extension.

The network of transcription factors is dynamically reorganized during the transition from naïve to formative pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that POU5F1 (also known as OCT4) and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation in naïve embryonic stem cells (ESCs). Loss of these enhancers attenuates the upregulation of Prdm14, leading to reduced Pramel7 induction and impaired degradation of UHRF1, which consequently diminished global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in a delayed transition from the naïve to formative pluripotent state. Our findings reveal that rodent-specific enhancer insertions endow Prdm14 with a dynamic regulatory architecture, enabling both activation and repression that collectively ensure the timely exit from naïve pluripotency during early embryogenesis.

The kidney is a complex organ requiring tightly coordinated interactions between epithelial, endothelial, and mesenchymal cells during development. Congenital kidney defects can result in kidney disease and renal failure, highlighting the importance of understanding kidney formation mechanisms. Advances in RNA sequencing have revealed remarkable cellular heterogeneity, especially in the kidney stroma, though relationships between stromal, epithelial, and endothelial cells remain unclear. This study presents a comprehensive gene expression atlas of embryonic and postnatal kidneys, integrating single-nucleus and in situ RNA sequencing data. We developed the Kidney Spatial Transcriptome Analysis Tool (KSTAT), enabling researchers to identify cell locations, predict cell-cell communication, and map gene pathway activity. Using KSTAT, we were able to uncover significant heterogeneity among embryonic kidney pericytes, providing a critical resource for hypothesis generation and advancing knowledge of kidney development and disease.

Reactive oxygen species (ROS) are crucial for wound healing and regeneration in several animals. In their work, Tetsuya Bando and colleagues show that Duox-derived ROS play a regulatory role during leg regeneration in cricket. To learn about their work, we spoke to the first author, Misa Okumura-Hirono, and the corresponding author, Tetsuya Bando, Senior Assistant Professor, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan.

Gastruloids are embryonic stem cell-derived in vitro models that mimic the axial patterning of the gastrulating embryo. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27), with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In their recent publication, Balayo et al. have investigated whether these formulations result in significant differences in gastruloid development. To find out more about their work, we spoke to first author Tina Balayo, and to co-corresponding authors André Dias and David Turner. In the Barcelona team, Tina and André are members of the Martínez Arias lab at the Universitat Pompeu Fabra (Barcelona, Spain), where Tina works as a research technician and lab manager, and André is an EMBO postdoctoral fellow. Heading the Liverpool team, David is a lecturer and group leader of the Development and Stem Cell Lab at the University of Liverpool, UK.

To generate haploid gametes, germ cells must transition from mitosis to meiosis. In mammals, transcriptional activator STRA8-MEIOSIN mediates the decision to enter the meiotic cell cycle, but how germ cells prevent continued mitotic cycling before meiotic entry remains unclear. MEIOC was previously shown to repress the mitotic program after meiotic entry. Here, we investigate MEIOC's role in the mitosis-to-meiosis transition during mouse oogenesis. Using cell proliferation analysis and cell cycle transcriptomics, we demonstrate that MEIOC prevents continued mitotic cycling prior to meiotic entry in oogenic cells. We find that G1/S cyclin CCNA2 is downregulated during the mitosis-to-meiosis transition, and MEIOC contributes to this downregulation. MEIOC also promotes entry into meiosis by increasing Meiosin transcript abundance and consequently activating STRA8-MEIOSIN. We also demonstrate that BMP signaling halts mitotic cycling and promotes meiotic entry by upregulating MEIOC. Thus, in mouse oogenic cells, the transition from mitosis to meiosis occurs as two molecularly regulated steps- (i) halt of mitotic cycling and (ii) entry into the meiotic cell cycle - and that MEIOC modifies the cell cycle program to facilitate both steps in this transition.

Drosophila insulin-like peptide 8 (ILP8) is expressed in mature follicle cells, but not in younger follicles. In their work, Jianjun Sun and colleagues show that ILP8 is required to induce ovulation and egg laying in virgin Drosophila females. To learn more about their work, we spoke to the first author, Rebecca Oramas, and the corresponding author, Jianjun Sun, Professor of Physiology & Neurobiology, University of Connecticut, USA.

Understanding the molecular mechanisms driving lineage decisions and differentiation during development is challenging in complex systems with a diverse progenitor pool, such as the mammalian cerebellum. Importantly, how different transcription factors cooperate to generate neural diversity and the gene regulatory mechanisms that drive neuron production, especially during the late stages of cerebellum development, are poorly understood. We used single cell RNA-sequencing (scRNA-seq) to investigate the developmental trajectories of Nestin-expressing progenitors (NEPs) in the neonatal mouse cerebellum. We identified FOXO1 as a key regulator of NEP-to-inhibitory neuron differentiation, acting directly downstream of ASCL1. Genome occupancy and functional experiments using primary NEP cultures showed that both ASCL1 and FOXO1 regulate neurogenesis genes during differentiation while independently regulating proliferation and survival, respectively. Furthermore, we demonstrated that WNT signalling promotes the transition from an ASCL1+ to a FOXO1+ cellular state. Finally, the role of WNT signalling in promoting neuron production via FOXO1 is conserved in primary human NEP cultures. By resolving how cerebellar inhibitory neurons differentiate, our findings could have implications for cerebellar disorders such as spinocerebellar ataxia, where these cells are overproduced.

Gastruloids are 3D aggregates of pluripotent stem cells grown in suspension culture that mimic many aspects of gastrulation and early axial elongation. The N2B27 basal medium in which mouse gastruloids are cultured can either be home-made (HM-N2B27) with materials of known origin, or commercially sourced (NDiff227), where the exact formulation is unknown. In this study, we examined whether these formulations resulted in significant differences in gastruloid development. Our results reveal that while both media enable standard gastruloid elongation, HM-N2B27 gastruloids initiate the elongation process earlier, have a higher number of cells and an increased anterior domain. Despite the maintenance of overall gene expression patterns, RNAseq analysis indicated differences in cell fate specification, with HM-N2B27 gastruloids exhibiting higher expression of spinal cord-related genes, while NDiff227 favours mesodermal differentiation. Furthermore, differential gene enrichment analysis suggests that changes in key signalling pathways underlie the differences between HM-N2B27 and NDiff227 gastruloids. These findings highlight the importance of basal media composition for gastruloid development, underscoring the need for careful media selection during in vitro engineering of stem cell-based embryo models.

Typically, embryonic development is a continuous process. However, in some species, embryos can halt their development and enter a dormant state known as diapause. During this period, the embryo retains its viability and developmental capacity to resume transient embryogenesis. While the diapausing embryo appears to be in a state of suspended animation, recent studies have revealed a more dynamic picture of modulated signalling responses, metabolic rewiring and slow-paced but active tissue morphogenesis. In this Spotlight, we discuss the emerging concepts of the molecular and cellular mechanisms that govern mammalian embryonic diapause, focusing on the mouse as a model system.

Flowers are a key reproductive innovation of the angiosperms. Seed plant reproductive axes (including flowers) evolved as reproductively specialized shoots of the land plant diploid sporophyte, with the gamete-producing haploid gametophyte becoming reduced and enclosed within ovules and microsporangia. The transcription factor LEAFY (LFY) initiates floral development, yet it predates flowers and is found across all land plants. LFY function outside angiosperms is known from the moss Physcomitrium patens, where it controls the first cell division of the sporophyte, and from the model fern Ceratopteris richardii, a seedless vascular plant where CrLFY1 and CrLFY2 maintain vegetative meristem activity. However, how LFY's floral role evolved remains unclear. Using over-expression, we uncover new roles for CrLFY1/2 in fern gametophyte reproduction, in sperm cells and in the gametophyte's multicellular notch meristem. While no sporophytic reproductive function was detected in terms of time to sporing, over-expression supports a role in frond compounding and in the zygote's first cell division. Our findings suggest a potentially ancestral LFY function in fern haploid-stage reproduction, which might have been co-opted into the sporophyte during the origin of the flower.

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) resets the aging clock. However, primed iPSCs can retain cell-of-origin epigenomic marks, especially those linked to heterochromatin. Here we show that iPSCs produced from fibroblasts of late-onset sporadic Alzheimer's disease (AD) cases retain epigenomic alterations that correlate with developmental anomalies and neurodegeneration. When compared to controls, AD iPSCs show reduced BMI1 expression and H3K9me3 levels and an altered DNA methylome. Gene Ontology analysis of differentially methylated DNA regions (DMRs) reveals terms linked to cell-cell adhesion and synapse, with MEF2C binding sites being the most enriched at DMRs. Upon noggin exposure, AD iPSCs show lesser efficient neural induction and forebrain specification, together with elevated WNT signaling. Mature AD neurons present a mixed cell lineage identity phenotype and reduced MEF2C expression. AD glial cells express neuronal, cell proliferation, and stem cell-related genes. Despite these anomalies, AD iPSCs generate cortical neurons in normal proportion and readily form cerebral organoids showing AD-related pathologies. These findings implicate reprogramming resistant epigenomic alterations or genetic variants working in trans on the epigenome in AD pathophysiology.

The striped expression of pair-rule-genes in Drosophila embryos is a paradigm for understanding transcriptional control of development. Pair-rule-striped expression is regulated by two types of cis-regulatory elements: stripe-specific elements respond to non-periodic cues in different regions of the embryo to establish individual stripes while 7-stripe elements simultaneously regulate all stripes, responding to pair-rule-genes expressed in stripes. Here, we assess roles of stripe-specific versus 7-stripe elements for the pair-rule-gene ftz. We show that loss of a ftz stripe 2 element is compensated by 7-stripe elements, even though they respond to different spatiotemporal cues. We next ask if similar rules apply to the classic eve stripe2 element. Animals homozygous for a genomic deletion of eve stripe2 are viable and fertile; stripe 2 expression is perturbed early but re-establishes sufficiently to regulate downstream target genes. However, temperature or genetic stress decreases viability of ftz and eve stripe2 deletion mutants. Thus, these stripe-specific elements contribute to robustness but are not absolutely required for segment formation. Two separate routes to establishing stripes, stripe-specific and 7-stripe elements, buffer each other, adding complexity to embryonic patterning.

All exposed epithelial surfaces, including the walls of internal tubes, are lined by a lipid and glycoprotein-rich apical extracellular matrix (aECM) that helps shape and protect the apical domain. Secreted lipocalins are lipid transporters frequently found within apical compartments. We show that loss of the C. elegans lipocalin LPR-1 disrupts the assembly of another lipocalin, LPR-3, within the pre-cuticle aECM that protects and shapes the narrow excretory duct and pore tubes. Loss of SCAV-2, a CD36 family scavenger receptor, restored LPR-3 matrix localization and suppressed the tube shaping defects of lpr-1 and a subset of pre-cuticle mutants, but not lpr-3 mutants. SCAV-2 accumulates at duct and pore apical surfaces and functions locally within these tubes. These data demonstrate that LPR-1 and SCAV-2 have opposing effects on narrow tube integrity by altering the content and organization of that tube's luminal aECM, possibly by acting as transporters of LPR-3 or an LPR-3 cofactor. These results have broadly relevant implications regarding the importance of lipocalins and scavenger receptors for aECM organization and integrity of the narrowest tubes in the body.

Formation of the amnion in humans is crucial for fetal development and a healthy pregnancy. In addition to providing a protective layer to the developing fetus as a component of the amniochorionic fetal membranes, the amnion serves as a signaling center for patterning early embryonic tissues. However, because the amnion is first specified in the human epiblast during implantation, the molecular and cellular events of this early amniogenic process in humans cannot be studied in utero. Recent developments using new human stem cell-derived model systems, as well as single-cell and spatial transcriptomic analyses of early human and monkey embryos, have uncovered new insights into the underpinnings of primate amnion specification. Here, we highlight recent findings from human and monkey models with an emphasis on current understandings of morphogenesis, BMP-driven transcriptional signatures and key players associated with primate amniotic ectoderm specification.

During mammalian eye development, Sox2 is involved in the gene regulatory network that helps retinal progenitor cells (RPCs) gain neural competence, but how it mechanistically contributes to this process is unclear. In a new study, Issam Al Diri and colleagues address this and unravel the chromatin-based functions of SOX2 in the developing retina. To learn more about this work and the people behind it, we talked to first authors Fuyun Bian, Kimiasadat Golestaneh, Emily Davis and Abdullah Khan from the Retinal Epigenetics and Genomics Laboratory at the UPMC Vision Institute, University of Pittsburgh, PA, USA.