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bioRxiv2018

Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

MicroRNAs are a group of short non-coding RNAs that undertake various roles in different cell signaling pathways and developmental stages. They regulate gene expression levels at the post-transcriptional stage, which results in cleavage of mRNAs or repression of their translation. Some miRNAs, including the miR-302 cluster, are critical regulators for the stemness state of embryonic stem cells and cell fate patterning. The miR-302 cluster is located in the intron of a non-coding gene that has no other reported function, other than hosting miR-302, and grant a complex expression regulation through upstream its regulatory sequences. To date, analysis of the miR-302 expression pattern in a transgenic mouse model has not been reported. In this study, we generated transgenic mice that expressed EGFP driven by miR-302 upstream regulatory sequences that harbored the core promoter of its host gene. We examined the activity of the miR-302 promotor in somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that miR-302 highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and has low expression in the somatic tissues of transgenic mice. It could be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages in mice.

developmental biology↗

bioRxiv2018

Ask1 and Akt act synergistically to promote ROS-dependent regeneration in Drosophila

The mechanism by which apoptotic cells release signals that induce undamaged neighbor cells to proliferate and regenerate missing parts remains elusive. Oxidative stress originated by dying or damaged cells can be propagated to neighboring cells, which then promote regeneration. We investigated the nature of the stress sensing mechanism by which neighboring cells are recruited. We found that Drosophila apoptosis signal-regulating kinase 1 (Ask1) senses reactive oxygen species (ROS) differently in stressed dying cells and unstressed neighboring cells and this differential sensing is pivotal for tissue repair. In undamaged cells, this activity is attenuated, but not abolished, by Akt1 phosphorylation, which thus acts as a survival signal that results in the tolerable levels of p38 and JNK necessary for regeneration. These observations demonstrate that the non-autonomous activation of the ROS-sensing mechanism by Ask1 and Akt1 in neighboring unstressed cells. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration.\n\nAuthor summaryOne of the early events that occur after tissue damage is oxidative stress production that signals to initiate wound healing and regeneration. Several signaling pathways, such as JNK and p38, respond to oxidative stress and are necessary for regeneration. We decided to explore the mechanism that links the oxidative stress and the activation of these pathways. We used epithelia of Drosophila to genetically direct cell death in specific zones of the tissue as means of experimentally controlled cell damage. We found that the Ask1 protein, which is sensitive to oxidative stress, is a key player in this scenario. Actually it acts as an intracellular sensor that upon damage activates those signaling pathways. However, high activity of Ask1 can be toxic for the cell. This is controlled by Akt, an enzyme dowstream the insulin pathway, with attenuates the activity of Ask1 to tolerable levels. In conclusion, Ask1 and Akt act synergistically to respond to the stress generated after tissue damage and drive regeneration. In other words, we found that the link between oxidative stress and nutrition is key for tissue regeneration.

developmental biology↗

bioRxiv2018

Transitions in cell potency during early mouse development are driven by Notch

The Notch signalling pathway plays fundamental roles in diverse developmental processes in metazoans, where it is important in driving cell fate and directing differentiation of various cell types. However, we still have limited knowledge about the role of Notch in early preimplantation stages of mammalian development, or how it interacts with other signalling pathways active at these stages such as Hippo. By using genetic and pharmacological tools in vivo, together with image analysis of single embryos and pluripotent cell culture, we have found that Notch is active from the 4-cell stage. Transcriptomic analysis in single morula identified novel Notch targets, such as early naive pluripotency markers or transcriptional repressors such as TLE4. Our results reveal a previously undescribed role for Notch in driving transitions during the gradual loss of potency that takes place in the early mouse embryo prior to the first lineage decisions.

developmental biology↗

bioRxiv2018

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, but neither loss nor gain of hmx3a function was sufficient to generate ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.

developmental biology↗

bioRxiv2018

Nuclear export in somatic cyst cells controls cyst cell-germline coordination and germline differentiation in the Drosophila testis

Nucleocytoplasmic communication is crucial for proper cell function and coordination of intrinsic cues with signaling responses emanating from the neighboring cells and the local tissue microenvironment. In the Drosophila male germline system, germ cells proliferate and progressively differentiate enclosed in supportive somatic cyst cells, forming a small cyst, the functional unit of differentiation. Here we show that the peripheral nucleoporins Nup62, Nup214 and Nup88, and the exportin Emb are critically required in cyst cells to maintain cyst cell survival and germline encapsulation in order to protect cyst cell-germline communication and promote germ cell differentiation. Knockdown of nup62, emb, nup214 or nup88 in cyst cells leads to cell-autonomous defects in mRNA export, and cell non-autonomous overproliferation of early germ cells in the absence of cyst cell-derived differentiation signals. Suppression of apoptosis can reverse cyst cell elimination and partially restored those defects. Interestingly, overexpression of the Drosophila Profilin gene chickadee can rescue cyst cell survival and restore germline encapsulation and differentiation, by counteracting Ntf-2 mediated export, suggesting that the function of Profilin in cyst cells is linked to nuclear export.

developmental biology↗

bioRxiv2018

tbx5 and tbx15 mediate zebrafish posterior lateral line migration and development via a negative feedback loop

The zebrafish posterior lateral line (pLL) is a mechanosensory organ enabling detection of movement in the aqueous environment. Evidence suggests that two T-box transcription factors, tbx5 and tbx15, participate in pLL migration and development. Lateral line migration and deposition defects are observed following tbx5 or tbx15 morpholino knockdown. Additional studies demonstrate that the tbx5 phenotype is partially rescued when tbx15 is also knocked down. pLL defects similar to those of tbx5 are noted following knockdown of camKII-{beta}2, a known downstream target of Tbx5, suggesting a potential mechanistic pathway. Ectopic expression of human CamKII-{partial}C partially rescues the tbx5 phenotype but exacerbates the tbx15 phenotype. These results, combined with in situ hybridization and qRT-PCR profiling, indicate a negative feedback loop controlling multiple primordium patterning markers, including cxcr4b, cxcr7b, fgf10a and notch3, which ultimately affects pLL neuromast migration and deposition.

developmental biology↗

bioRxiv2018

Anti-tumor effects of antimicrobial peptides, targets of the innate immune system, against hematopoietic tumors in Drosophila mxc mutants

The innate immune response is the first line of defense against microbial infections. In Drosophila, three immune pathways induce the synthesis of antimicrobial peptides (AMPs) in the fat body. Recently, it has been reported that certain cationic AMPs exhibit selective cytotoxicity against human cancer cells. However, little is known about their anti-tumor effects. Drosophila mxcmbn1 mutants exhibit malignant hyperplasia in a larval hematopoietic organ called the lymph gland (LG). Here, using RNA-Seq analysis, we found that many immunoresponsive genes, including AMP genes, were up-regulated in the mutants. Down-regulation of these pathways by either a Toll or an imd mutation enhanced the tumor phenotype of the mxc mutants. Conversely, ectopic expression of each of five different AMPs in the fat body significantly suppressed the LG hyperplasia phenotype in the mutants. Thus, we propose that the Drosophila innate immune system can suppress progression of hematopoietic tumors by inducing AMP gene expression. Overexpression of any one of these five AMPs resulted in enhanced apoptosis in the mutant LGs, while no apoptosis signals were detected in controls. We observed that two AMPs, Drosomycin and Defensin, were taken up by circulating hemocyte-like cells, which were associated with LG regions showing reduced cell-to-cell adhesion in the mutants; another AMP, diptericin, was directly localized on the tumors without intermediating hemocytes. These results lead us to conclude that the AMPs have a specific cytotoxic effect that enhance apoptosis exclusively in the tumor cells.\n\nSummary statementAntimicrobial peptides can be associated with tumor cells generated in a hematopoietic tissue in Drosophila mxc mutants and have an anti-tumor effect in suppressing their growth.

developmental biology↗

bioRxiv2018

Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning

The generation of patterns during development is generally viewed as a direct process. In the mouse jaw, however, the sequential patterning of molars initiates with abortive tooth signaling centers called MS and R2, thought to be vestiges of the lost rodent premolars. Moreover, the mature signaling center of the first molar (M1) is formed from the fusion of two signaling centers (R2 and early M1). Here, we report that Edar expression reveals the hidden dynamics of signalling centers patterning. First, Edar expression evidenced a hidden two-step patterning process that we modelled with a single activator-inhibitor pair: the epithelium is initially broadly activated, then activation becomes restricted in space to give rise to the signalling centers. Second, Edar expression unveils successive phases of pattern making and pattern erasing events, a phenomenon that we called a developmental palimpsest. MS is erased by a broad activation for the benefit of R2, which itself is erased before it recovers when the first molar signaling center forms. In the lower but not the upper jaw, the two neighboring signaling centers then fuse into a single elongated center. Our model recapitulated the erasure of the R2 signaling center by the wave of activation that precedes the formation of M1 signaling center, and predicted the surprising rescue of R2 in the context of an Edar mutant with reduced activation. It suggested that R2 was not intrinsically defective, but actively outcompeted by M1 formation. We confirmed this by cultivating R2 separately from the posterior tissue and showing it could then generate a tooth. Finally, by introducing chemotaxis as a secondary process of tooth germ maturation, we recapitulated the fusion of R2 and M1 in the lower jaw only, and the loss of fusion when Edar function is impaired in organ cultures. In conclusion, we have uncovered a highly indirect and dynamic nature of pattern formation in the molar field that could nevertheless be simulated with simple mathematical models. Our study argues for viewing embryonic patterns as dynamical objects rather than as fixed endpoints, where dynamics is essential to the outcome of the patterning process.

developmental biology↗

bioRxiv2018

fruitless functions downstream of doublesex to promote sexual dimorphism of the gonad stem cell niche

Backgrounddoublesex (dsx) and fruitless (fru) are the two downstream transcription factors that actuate Drosophila sex determination. While dsx assists fru to regulate sex-specific behavior, whether fru collaborates with dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs.\n\nResultsHere we report that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously studied regulation of sex-specific Fru expression, which is regulated by alternative splicing by Transformer (Tra), we show that male-specific expression of fru is regulated downstream of dsx, and is independent of Tra. Regulation of fru by dsx also occurs in the nervous system. fru genetically interacts with dsx to support maintenance of the hub throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, we identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the hub, indicating that fru is likely a direct target for transcriptional regulation by Dsx.\n\nConclusionsThese findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra.

developmental biology↗

bioRxiv2026

Distinct functions of Nup93 paralogs in tumor growth and Polycomb-mediated repression of JAK/STAT signaling

Nuclear pore complexes (NPCs) are nuclear envelope (NE)-embedded protein assemblies that mediate nucleocytoplasmic exchange and interact with the genome, including binding of an NPC component Nup93 to Polycomb chromatin domains. Here, we investigated the in vivo relevance of this relationship in Drosophila, which unusually contains two distinct paralogs of Nup93. Interestingly, we identified a Nup93-2-specific tumorigenic phenotype in larval wings, where depletion of Nup93-2, but not Nup93-1, led to tumor-like overgrowth, reminiscent of Polycomb mutations. Consistently, our transcriptomic analysis revealed a wide-spread loss of gene silencing in Nup93-2-depleted wings, particularly in a Nup93-bound Polycomb domain spanning genes for activators of JAK/STAT signaling. Nup93 paralogs were not found to differ in their effect on NPC biogenesis but strikingly, showed differences in subnuclear localization patterns. While Nup93-1 co-localized exclusively with fully assembled NPCs, Nup93-2 exhibited only partial co-localization and was found at additional NE locations in a tissue-specific manner. Together, our results identify an in vivo silencing role of a Nup93 paralog and suggest that Nup93-2 may form a unique NE-associated complex that targets a subset of Polycomb domains containing growth-promoting genes.

developmental biology↗

bioRxiv2026

miR-34/449 miRNAs regulate choroid plexus ciliogenesis to control cerebrospinal fluid production

A developmental increase in cerebrospinal fluid (CSF) production during development is essential for neuronal growth and ventricular expansion. A key regulator of CSF production is the specialized sensory multicilia of the choroid plexus (ChP), which mediate non-canonical Sonic hedgehog (Shh) signaling to suppress water channel and ion transporter expression, thereby limiting CSF production. ChP multicilia progressively shortens during development, attenuating Shh signaling and promoting CSF production. Here, we identify miR-34/449 miRNAs as essential regulators of ChP multiciliogenesis. Whereas mutations in canonical ciliogenesis genes elevate CSF production and contribute to hydrocephaly, deletion of miR-34/449 reduces CSF volume and causes microcephaly. Loss of miR-34/449 miRNAs causes excessive basal body amplification, defective basal body docking, and failure of developmental multiciliary shortening. Consequently, miR-34/449-deficient ChP cilia remain abnormally long and fail to attenuate Shh signaling, resulting in sustained repression of water channel and ion transporter expression and reduced CSF production. Mechanistically, miR-34/449 miRNAs directly target Gmnc, a master transcriptional regulator of multiciliogenesis, to restrain basal body amplification and promote basal body docking. Together, our findings identify miR-34/449 miRNAs as critical regulators of ChP multiciliogenesis and establish the developmental remodeling of ChP multicilia as a mechanism to couple Shh signaling dynamics to developmental control of CSF production.

developmental biology↗

bioRxiv2026

Function-driven geometry directs human pilosebaceous unit development

Single-cell technologies have generated cell censuses of tissues, however, how tissue geometry reflects functional needs remains poorly characterized. The human pilosebaceous unit offers a tractable model, a prenatally-formed complex mini-organ combining hair and sebum production with a stem cell reservoir. Using histomorphology, spatial transcriptomics, and single-cell multiomics on the same human prenatal scalp skin samples (8-19 post-conception weeks), integrated and analyzed using machine learning approaches, we built a spatiotemporal map of pilosebaceous unit development. We demonstrate that epithelial-mesenchymal interactions coordinate cellular fate and organogenesis, using an in vitro hair-bearing skin organoid model to validate this tissue-patterning. In addition, we show sebaceous gland developmental programmes are overcome during tumor formation. Our large-scale multi-modal analysis provides a unique framework for understanding form and function of tissues with applications in tissue engineering and pathology.

developmental biology↗

bioRxiv2026

Injury size regulates glucose allocation locally and systemically during vertebrate tissue regeneration

Tissue regeneration requires careful allocation of metabolic resources, yet how organisms adjust this allocation in response to varying amounts of tissue loss remains poorly understood. Here, we show that the regenerative metabolic response is not fixed: the size of an injury regulates how glucose is allocated at both local and organism-wide levels. We first demonstrate that tail regeneration requires glucose metabolism in the axolotl (Ambystoma mexicanum), a salamander capable of regenerating centimetre-scale tissues. We then mapped glucose uptake in axolotls regenerating from small or large tail injuries using positron emission tomography/magnetic resonance imaging (PET/MRI) and the radiolabelled glucose analogue [18F]FDG. Glucose uptake was elevated in regenerating tails compared to uninjured tails. During early regeneration, larger injuries induced higher glucose uptake than smaller injuries, correlating with faster regenerative outgrowth. Larger injuries also increased glucose uptake in distant organs, indicating a systemic metabolic response. Together, our findings suggest that metabolic responses tuned to injury size underlie faithful tissue regeneration and establish PET/MRI as a powerful approach for studying whole-body metabolic dynamics in large regenerating vertebrates.

developmental biology↗

bioRxiv2026

An ancestral pronephric contribution reveals the multilineage origin of the teleost gonad and revises the evolution of vertebrate gonadogenesis

Challenging the paradigm that pronephric field contribution to gonadal formation would be an amniote innovation, we demonstrate this trait is ancestral to bony vertebrates. Using cell lineage tracing, single-cell and spatial transcriptomics, and functional validation, we show that the teleost gonad arises from three distinct embryonic tissues, the pronephros, the coelomic epithelium, and the lateral plate mesoderm, in contrast to amniotes. This multi-tissue origin generates an unexpected lineage-based cellular diversity. Further cross-species comparisons over medaka, mouse, chicken and turtle unravel how lineage-specific deviations shape early gonadal development. Specifically, we map these variations amongst the different gene regulatory networks, outlining their physiological implications for specialized gonadal functions. Our results support a model in which heterochronic shifts are coupled to regulatory rewiring of conserved gene networks, driving lineage-specific developmental trajectories through a canalized developmental system drift.

developmental biology↗

bioRxiv2026

GDNF enemas improve epithelial and immune defects in both aganglionic and ganglionic colon of Hirschsprung mice

Hirschsprung disease (HSCR) is a severe birth defect where ganglia of the enteric nervous system (ENS) are missing from distal bowel. The aganglionic segment is also characterized by increased epithelial permeability and pro-inflammatory immune activation. These problems may sequentially lead to translocation of gut microbes into the colon wall and systemic circulation, resulting in enterocolitis and sepsis. Current HSCR treatment via surgical resection of the aganglionic segment is lifesaving but not curative, often leaving patients with persistent gastrointestinal complications including recurrent risk of enterocolitis. As alternative, we are developing a regenerative medicine strategy based on in situ stimulation of tissue-resident ENS progenitors via rectal administration of the neurotrophic factor GDNF. Here, we report that GDNF-based therapy has pleiotropic gastrointestinal effects in a mouse model of short-segment HSCR, beyond its role in ENS regeneration. Interestingly, we found that these protective effects are not restricted to the aganglionic distal colon, also positively impacting the ENS-containing proximal colon. GDNF treatment reduces bacterial translocation both locally and in peripheral organs, and this is associated with recovery of the key epithelial junction proteins CLDN3, ZO1 and DSG2. Furthermore, multiparameter flow cytometry-based analysis of 55 lymphoid and 17 myeloid cell subtypes revealed that GDNF treatment has global anti-inflammatory effects, preferentially affecting innate over adaptive immunity. Overall, these findings highlight a critical role for GDNF treatment in reestablishing proper epithelial and immune cell homeostasis, offering promising therapeutic avenues not only for HSCR but also potentially for other intestinal disorders with overlapping pathophysiology.

developmental biology↗

bioRxiv2026

Neogenin-1 marks myeloid-primed fetal hematopoietic stem cells that undergo progressive lineage-restriction with age

During aging, hematopoietic stem cells (HSCs) increasingly shift from balanced to myeloid-biased differentiation, resulting in reduced lymphoid output and impaired adaptive immunity. The question of whether this lineage bias is established in a subset of HSCs during early development or primarily emerges with aging warrants further investigation. Here, we investigate whether myeloid-biased HSCs (my-HSCs) are established at the fetal liver stage by specifically examining Neogenin-1 (NEO1), a previously defined marker of my-HSCs. We identify two distinct populations of Hoxb5+ HSCs in the fetal liver: NEO1+ and NEO1-, with NEO1+ HSCs exhibiting transcriptional and functional characteristics consistent with my-HSCs. With age, my-HSC-associated transcriptional programs become increasingly reinforced across the Hoxb5+ pHSC compartment, with NEO1+ cells showing early enrichment of this program and both NEO1+ and NEO1- cells acquiring broader myeloid-biased features in aging. These findings suggest that lineage programming can begin early in development and is further shaped by age-related changes, potentially contributing to the functional decline observed in the aging hematopoietic system.

developmental biology↗

bioRxiv2026

Dynamic coupling of cell fate specification and cell sorting during mouse preimplantation development

During preimplantation development in mice, cells of the inner cell mass undergo a cell fate decision to become either Epiblast (Epi) or Primitive Endoderm (PrE) cells. Cell fate patterns during this stage range from an alternating pattern at the beginning to the separation of Epi and PrE at the end. Several mechanisms guiding this decision and pattern formation have been proposed, including intra- and intercellular signalling, cell division and cell sorting. The current understanding is that signalling generates the cell fates and subsequent sorting introduces the spatial cell fate separation. We used agent-based modelling to investigate whether cell differentiation and cell sorting can act concurrently and how their relative contributions to pattern formation may change over time. Comparing our model to experimental data for mouse blastocysts and ICM organoids, we find two mechanistic regimes that can produce the experimentally observed spatial separation: (i) simultaneous long-range intercellular signalling and cell sorting, and (ii) a gradual transition from short-range signalling to cell sorting, in which the timing is mediated via reducing cell fate plasticity. While the second agrees better with existing experimental evidence for late blastocysts, the first might still be relevant for early and mid blastocysts. Together, our results refine the sequential view of Epi/PrE patterning by showing that fate specification and cell sorting can be dynamically coupled, with their relative contributions changing over the course of blastocyst development.

developmental biology↗

bioRxiv2026

Distinct roles for partially redundant transcription factors in Caenorhabditis elegans mesoderm lineage development

Developmental transcription factors often have overlapping functions, making it difficult to define the distinct roles of individual factors during lineage specification. We investigated the partially redundant transcription factors TBX-35 and CEH-51 in the Caenorhabditis elegans embryonic MS mesodermal lineage using 4D lineage tracing, reporter imaging, genetics, and single-cell RNA sequencing. In tbx-35 mutants, MS descendants showed progressively slower cell cycles and a division pattern that increasingly resembled the cousin C lineage. Fate-regulator expression also shifted toward C-like features, including ectopic pal-1 and expanded HLH-1 expression, although mutant cells did not simply adopt normal C-lineage positions. Loss of tbx-35 also impaired a later MS-dependent Notch induction in the AB lineage while leaving an earlier induction intact. CEH-51 showed a different pattern of activity whereby its protein became enriched in anterior MS daughters, and ceh-51 mutants produced later, more restricted lineage defects that were strongest in descendants of cells with higher CEH-51 levels. Single-cell profiling identified overlapping but nonidentical sets of genes dependent on the two factors. TBX-35-dependent changes were strongest at earlier stages, whereas CEH-51-dependent genes became more prominent later and were enriched in anterior MS sublineages. Finally, temperature-shift experiments determined that the severity and onset of tbx-35 mutant phenotypes depend on the maternal temperature environment and cannot be explained by differences in residual CEH-51 expression. These findings reveal that TBX-35 and CEH-51 contribute differently across the MS lineage and that reliable mesoderm development is supported by overlapping zygotic and maternal regulatory inputs.

developmental biology↗