A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Engineered Wnt7a ligands rescue blood-brain barrier and cognitive deficits in a COVID-19 mouse model.

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating cerebrovascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we performed unbiased transcriptional analysis to identify brain endothelial cell signalling pathways dysregulated by mouse adapted SARS-CoV-2 MA10 in aged immunocompetent C57Bl/6 mice in vivo. This analysis revealed significant suppression of Wnt/ß-catenin signalling, a critical regulator of blood-brain barrier (BBB) integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/ß-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected BBB integrity, reduced T-cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/ß-catenin signalling or its downstream effectors could be potential interventional strategies for restoring cognitive health following viral infections.

The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish.

BACKGROUND: Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. RESULTS: We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. CONCLUSIONS: Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.

EVL regulates VEGF receptor-2 internalization and signaling in developmental angiogenesis.

Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.

Biallelic mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence.

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

Ezrin Is a Novel Protein Partner of Aquaporin-5 in Human Salivary Glands and Shows Altered Expression and Cellular Localization in Sjögren's Syndrome.

Sjögren's syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly distributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5-ezrin interaction was assessed by immunoprecipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5-ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunoreactivity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5-ezrin co-localization. Our data reveal that AQP5-ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.

Defective adgra2 (gpr124) splicing and function in zebrafish ouchless mutants.

A hitherto unidentified N-ethyl-N-nitrosourea (ENU)-induced mutation affects dorsal root ganglia (DRG) formation in ouchless mutant zebrafish larvae. In contrast to previous findings assigning the ouchless phenotypes to downregulated sorbs3 transcript levels, this work re-attributes the phenotypes to an essential splice site mutation affecting adgra2 (gpr124) splicing and function. Accordingly, ouchless mutants fail to complement previously characterized adgra2 mutants and exhibit highly penetrant cerebrovascular defects. The aberrantly spliced adgra2 transcript found in ouchless mutants encodes a receptor lacking a single leucine-rich repeat (LRR) within its N-terminus.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Mechanism of Trypanosoma brucei gambiense resistance to human serum.

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

The Trypanosoma brucei TbHrg protein is a heme transporter involved in the regulation of stage-specific morphological transitions.

The human parasite Trypanosoma brucei does not synthesize heme de novo and instead relies entirely on heme supplied by its vertebrate host or its insect vector, the tsetse fly. In the host bloodstream T. brucei scavenges heme via haptoglobin-hemoglobin (HpHb) receptor-mediated endocytosis occurring in the flagellar pocket. However, in the procyclic developmental stage, in which T. brucei is confined to the tsetse fly midgut, this receptor is apparently not expressed, suggesting that T. brucei takes up heme by a different, unknown route. To define this alternative route, we functionally characterized heme transporter TbHrg in the procyclic stage. RNAi-induced down-regulation of TbHrg in heme-limited culture conditions resulted in slower proliferation, decreased cellular heme, and marked changes in cellular morphology so that the cells resemble mesocyclic trypomastigotes. Nevertheless, the TbHrg KO developed normally in the tsetse flies at rates comparable with wild-type cells. T. brucei cells overexpressing TbHrg displayed up-regulation of the early procyclin GPEET and down-regulation of the late procyclin EP1, two proteins coating the T. brucei surface in the procyclic stage. Light microscopy of immunostained TbHrg indicated localization to the flagellar membrane, and scanning electron microscopy revealed more intense TbHrg accumulation toward the flagellar pocket. Based on these findings, we postulate that T. brucei senses heme levels via the flagellar TbHrg protein. Heme deprivation in the tsetse fly anterior midgut might represent an environmental stimulus involved in the transformation of this important human parasite, possibly through metabolic remodeling.

Apolipoproteins L control cell death triggered by TLR3/TRIF signaling in dendritic cells.

Apolipoproteins L (ApoLs) are Bcl-2-like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll-like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α(+)  dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter-inducing interferon ß) signaling pathway and is dependent on IFN-ß in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti-apoptotic protein Bcl-xL (where Bcl-xL is B-cell lymphoma extra large). Similarly, in human monocyte-derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3-like peptide of ApoLs appears to be involved in the DC death-promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.

Translational profiling through biotinylation of tagged ribosomes in zebrafish.

Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.

Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus.

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth.

Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24ß-methyltransferase (TbSMT) and sterol 14α-demethylase [TbSDM (TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of TbSMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 µM from lipid-depleted media) with small amounts of ergosterol (1.2 µM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanism-based inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 µM each (ED50 values) resulting in parasite death. Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection.

Experimental therapy of African trypanosomiasis with a nanobody-conjugated human trypanolytic factor.

High systemic drug toxicity and increasing prevalence of drug resistance hampers efficient treatment of human African trypanosomiasis (HAT). Hence, development of new highly specific trypanocidal drugs is necessary. Normal human serum (NHS) contains apolipoprotein L-I (apoL-I), which lyses African trypanosomes except resistant forms such as Trypanosoma brucei rhodesiense. T. b. rhodesiense expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT. A truncated apoL-I (Tr-apoL-I) has been engineered by deleting its SRA-interacting domain, which makes it lytic for T. b. rhodesiense. Here, we conjugated Tr-apoL-I with a single-domain antibody (nanobody) that efficiently targets conserved cryptic epitopes of the variant surface glycoprotein (VSG) of trypanosomes to generate a new manmade type of immunotoxin with potential for trypanosomiasis therapy. Treatment with this engineered conjugate resulted in clear curative and alleviating effects on acute and chronic infections of mice with both NHS-resistant and NHS-sensitive trypanosomes.

A new microfluidic model to study dendritic remodeling and mitochondrial dynamics during axonal regeneration of adult zebrafish retinal neurons.

Unlike mammals, adult zebrafish are able to fully regenerate axons and functionally recover from neuronal damage in the mature central nervous system (CNS). Decades of research have tried to identify the mechanisms behind their spontaneous regenerative capacity, but the exact underlying pathways and molecular drivers remain to be fully elucidated. By studying optic nerve injury-induced axonal regrowth of adult zebrafish retinal ganglion cells (RGCs), we previously reported transient dendritic shrinkage and changes in the distribution and morphology of mitochondria in the different neuronal compartments throughout the regenerative process. These data suggest that dendrite remodeling and temporary changes in mitochondrial dynamics contribute to effective axonal and dendritic repair upon optic nerve injury. To further elucidate these interactions, we here present a novel adult zebrafish microfluidic model in which we can demonstrate compartment-specific alterations in resource allocation in real-time at single neuron level. First, we developed a pioneering method that enables to isolate and culture adult zebrafish retinal neurons in a microfluidic setup. Notably, with this protocol, we report on a long-term adult primary neuronal culture with a high number of surviving and spontaneously outgrowing mature neurons, which was thus far only very limitedly described in literature. By performing time-lapse live cell imaging and kymographic analyses in this setup, we can explore changes in dendritic remodeling and mitochondrial motility during spontaneous axonal regeneration. This innovative model system will enable to discover how redirecting intraneuronal energy resources supports successful regeneration in the adult zebrafish CNS, and might facilitate the discovery of new therapeutic targets to promote neuronal repair in humans.

Local angiogenic interplay of Vegfc/d and Vegfa controls brain region-specific emergence of fenestrated capillaries.

Fenestrated and blood-brain barrier (BBB)-forming endothelial cells constitute major brain capillaries, and this vascular heterogeneity is crucial for region-specific neural function and brain homeostasis. How these capillary types emerge in a brain region-specific manner and subsequently establish intra-brain vascular heterogeneity remains unclear. Here, we performed a comparative analysis of vascularization across the zebrafish choroid plexuses (CPs), circumventricular organs (CVOs), and retinal choroid, and show common angiogenic mechanisms critical for fenestrated brain capillary formation. We found that zebrafish deficient for Gpr124, Reck, or Wnt7aa exhibit severely impaired BBB angiogenesis without any apparent defect in fenestrated capillary formation in the CPs, CVOs, and retinal choroid. Conversely, genetic loss of various Vegf combinations caused significant disruptions in Wnt7/Gpr124/Reck signaling-independent vascularization of these organs. The phenotypic variation and specificity revealed heterogeneous endothelial requirements for Vegfs-dependent angiogenesis during CP and CVO vascularization, identifying unexpected interplay of Vegfc/d and Vegfa in this process. Mechanistically, expression analysis and paracrine activity-deficient vegfc mutant characterization suggest that endothelial cells and non-neuronal specialized cell types present in the CPs and CVOs are major sources of Vegfs responsible for regionally restricted angiogenic interplay. Thus, brain region-specific presentations and interplay of Vegfc/d and Vegfa control emergence of fenestrated capillaries, providing insight into the mechanisms driving intra-brain vascular heterogeneity and fenestrated vessel formation in other organs.

Prdm12 represses the expression of the visceral neuron determinants Phox2a/b in developing somatosensory ganglia.

Prdm12 is a transcriptional regulator essential for the emergence of the somatic nociceptive lineage during sensory neurogenesis. The exact mechanisms by which Prdm12 promotes nociceptor development remain, however, poorly understood. Here, we report that the trigeminal and dorsal root ganglia hypoplasia induced by the loss of Prdm12 involves Bax-dependent apoptosis and that it is accompanied by the ectopic expression of the visceral sensory neuron determinants Phox2a and Phox2b, which is, however, not sufficient to impose a complete fate switch in surviving somatosensory neurons. Mechanistically, our data reveal that Prdm12 is required from somatosensory neural precursors to early post-mitotic differentiating nociceptive neurons to repress Phox2a/b and that its repressive function is context dependent. Together, these findings reveal that besides its essential role in nociceptor survival during development, Prdm12 also promotes nociceptor fate via an additional mechanism, by preventing precursors from engaging into an alternate Phox2 driven visceral neuronal type differentiation program.

An integrated model for Gpr124 function in Wnt7a/b signaling among vertebrates.

Within the central nervous system, Wnt7a/b are unambiguously discriminated from other Wnt ligands by an endothelial receptor complex made of the glycosylphosphatidylinositol (GPI)-anchored Reck and the adhesion G protein-coupled receptor (GPCR) Gpr124. Reck is a Wnt7a/b-specific receptor, while Gpr124 facilitates the delivery of Reck-bound Wnt7a/b ligands to Frizzled, through partially characterized mechanisms. We report that, in zebrafish, the Gpr124-Frizzled interactions are dominated by intracellular scaffolds that exploit the striking molecular mimicry between Gpr124 and Frizzled intracellular domains (ICDs): an internal Dvl-binding motif and a C-terminal ETTV motif that recruits Dlg4 and Magi3. By contrast, mammalian Gpr124 receptors exhibit an ICD-independent interaction mechanism governed by species-specific attributes of their transmembrane and extracellular domains. This mechanism seemingly evolved to replace the Dvl-mediated mechanism. By contrasting zebrafish, mouse, and human Gpr124, this study provides insights into the evolution of Gpr124/Reck function across the vertebrate clade, a receptor complex uniquely implicated in Wnt ligand-specific cellular responses.

Heme-deficient metabolism and impaired cellular differentiation as an evolutionary trade-off for human infectivity in Trypanosoma brucei gambiense.

Resistance to African trypanosomes in humans relies in part on the high affinity targeting of a trypanosome lytic factor 1 (TLF1) to a trypanosome haptoglobin-hemoglobin receptor (HpHbR). While TLF1 avoidance by the inactivation of HpHbR contributes to Trypanosoma brucei gambiense human infectivity, the evolutionary trade-off of this adaptation is unknown, as the physiological function of the receptor remains to be elucidated. Here we show that uptake of hemoglobin via HpHbR constitutes the sole heme import pathway in the trypanosome bloodstream stage. T. b. gambiense strains carrying the inactivating mutation in HpHbR, as well as genetically engineered T. b. brucei HpHbR knock-out lines show only trace levels of intracellular heme and lack hemoprotein-based enzymatic activities, thereby providing an uncommon example of aerobic parasitic proliferation in the absence of heme. We further show that HpHbR facilitates the developmental progression from proliferating long slender forms to cell cycle-arrested stumpy forms in T. b. brucei. Accordingly, T. b. gambiense was found to be poorly competent for slender-to-stumpy differentiation unless a functional HpHbR receptor derived from T. b. brucei was genetically restored. Altogether, we identify heme-deficient metabolism and disrupted cellular differentiation as two distinct HpHbR-dependent evolutionary trade-offs for T. b. gambiense human infectivity.