Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARL) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we used proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ∼3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely, SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

BACKGROUND: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. RESULTS: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. CONCLUSIONS: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state.

The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.

Polycomb Repressive Complexes in Hox Gene Regulation: Silencing and Beyond: The Functional Dynamics of Polycomb Repressive Complexes in Hox Gene Regulation.

The coordinated expression of the Hox gene family encoding transcription factors is critical for proper embryonic development and patterning. Major efforts have thus been dedicated to understanding mechanisms controlling Hox expression. In addition to the temporal and spatial sequential activation of Hox genes, proper embryonic development requires that Hox genes get differentially silenced in a cell-type specific manner as development proceeds. Factors contributing to Hox silencing include the polycomb repressive complexes (PRCs), which control gene expression through epigenetic modifications. This review focuses on PRC-dependent regulation of the Hox genes and is aimed at integrating the growing complexity of PRC functional properties in the context of Hox regulation. In particular, mechanisms underlying PRC binding dynamics as well as a series of studies that have revealed the impact of PRC on the 3D organization of the genome is discussed, which has a significant role on Hox regulation during development.

Multifaceted Hoxa13 function in urogenital development underlies the Hand-Foot-Genital Syndrome.

Hand-Foot-Genital syndrome is a rare condition caused by mutations in the HOXA13 gene and characterized by limb malformations and urogenital defects. While the role of Hoxa13 in limb development has been extensively studied, its function during the development of the urogenital system remains elusive mostly due to the embryonic lethality of Hoxa13 homozygous mutant mice. Using a conditional inactivation strategy, we show that mouse fetuses lacking Hoxa13 function develop megaureters, hydronephrosis and malformations of the uterus, reminiscent of the defects characterizing patients with Hand-Foot-Genital syndrome. Our analysis reveals that Hoxa13 plays a critical role in Müllerian ducts fusion and in ureter remodeling by regulating the elimination of the caudal common nephric duct, eventually preventing the separation from the nephric duct. Our data also reveal a specific role for Hoxa13 in the urogenital sinus, which is in part mediated by Gata3, as well as Hoxa13 requirement for the proper organization of the ureter. Finally, we provide evidence that Hoxa13 provides positional and temporal cues during the development of the lower urogenital system, a sine qua non condition for the proper function of the urinary system.

Insights on the role of hox genes in the emergence of the pentadactyl ground state.

Tetrapods are characterized by the presence of digits at the distal end of their limbs, which have emerged during the transition from fins to limbs. While variations in digit number are observed in extant tetrapods, most have five digits per limb and divergence from this pentadactyl ground state is always a reduction in digit number. Paleontological data revealed that stem-group tetrapods were polydactylous indicating that the evolution from fish fin to modern tetrapod limbs involved two major transitions; the emergence of digits and the shift from polydactyly to pentadactyly. The absence of living polydactyl tetrapod species is a major limitation in assessing the foundation of the pentadactyl constraint. Nonetheless, several genes having the capacity of modulating digit number have been identified and studying their functional and regulatory phylogeny will likely be critical in our comprehension of the emergence of the pentadactyl state. In this review, we provide an overview of the data obtained from mouse genetics that uncovered the role of Hox genes in controlling digit number and discuss regulatory changes that could have been implicated in the emergence of the pentadactyl ground state.

Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth.

Limb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hox-dependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes.

Clustering of tissue-specific sub-TADs accompanies the regulation of HoxA genes in developing limbs.

HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.

A Hoxa13:Cre mouse strain for conditional gene manipulation in developing limb, hindgut, and urogenital system.

The developing limb is a useful model for studying organogenesis and developmental processes. Although Cre alleles exist for conditional loss- or gain-of-function in limbs, Cre alleles targeting specific limb subdomains are desirable. Here we report on the generation of the Hoxa13:Cre line, in which the Cre gene is inserted in the endogenous Hoxa13 gene. We provide evidence that the Cre is active in embryonic tissues/regions where the endogenous Hoxa13 gene is expressed. Our results show that cells expressing Hoxa13 in developing limb buds contribute to the entire autopod (hand/feet) skeleton and validate Hoxa13 as a distal limb marker as far as the skeleton is concerned. In contrast, in the limb musculature, Cre-based fate mapping shows that almost all muscle masses of the zeugopod (forearm) and part of the triceps contain Hoxa13-expressing cells and/or their descendants. Besides the limb, the activity of the Cre is detectable in the urogenital system and the hindgut, primarily in the epithelium and smooth muscles. Together our data show that the Hoxa13:Cre allele is a useful tool for conditional gene manipulation in the urogenital system, posterior digestive tract, autopod and part of the limb musculature.

Recruitment of 5' Hoxa genes in the allantois is essential for proper extra-embryonic function in placental mammals.

The Hox gene family is well known for its functions in establishing morphological diversity along the anterior-posterior axis of developing embryos. In mammals, one of these genes, Hoxa13, is crucial for embryonic survival, as its function is required for the proper expansion of the fetal vasculature in the placenta. Thus, it appears that the developmental strategy specific to placental mammals is linked, at least in part, to the recruitment of Hoxa13 function in developing extra-embryonic tissues. Yet, the mechanism underlying this extra-embryonic recruitment is unknown. Here, we provide evidence that this functional novelty is not exclusive to Hoxa13 but is shared with its neighboring Hoxa11 and Hoxa10 genes. We show that the extra-embryonic function of these three Hoxa genes stems from their specific expression in the allantois, an extra-embryonic hallmark of amniote vertebrates. Interestingly, Hoxa10-13 expression in the allantois is conserved in chick embryos, which are non-placental amniotes, suggesting that the extra-embryonic recruitment of Hoxa10, Hoxa11 and Hoxa13 most likely arose in amniotes, i.e. prior to the emergence of placental mammals. Finally, using a series of targeted recombination and transgenic assays, we provide evidence that the regulatory mechanism underlying Hoxa expression in the allantois is extremely complex and relies on several cis-regulatory sequences.

"Self-regulation," a new facet of Hox genes' function.

BACKGROUND: Precise temporal and spatial expression of the clustered Hox genes is essential for patterning the developing embryo. Temporal activation of Hox genes was shown to be cluster-autonomous. However, gene clustering appears dispensable for spatial colinear expression. Generally, a set of Hox genes expressed in a group of cells instructs these cells about their fate such that the differential expression of Hox genes results in morphological diversity. The spatial colinearity is considered to rely both on local and long-range cis regulation. RESULTS: Here, we report on the global deregulation of HoxA and HoxD expression patterns upon inactivation of a subset of HOXA and HOXD proteins. CONCLUSIONS: Our data suggest the existence of a "self-regulation" mechanism, a process by which HOX proteins establish and/or maintain the spatial domains of the Hox gene family and we propose that the functionally dominant HOX proteins could contribute to generating the spatial parameters of Hox expression in a given tissue, i.e., HOX controlling the establishment of the ultimate HOX code.

Mapping the global interactome of the ARF family reveals spatial organization in cellular signaling pathways.

The ADP-ribosylation factors (ARFs) and ARF-like (ARLs) GTPases serve as essential molecular switches governing a wide array of cellular processes. In this study, we utilized proximity-dependent biotin identification (BioID) to comprehensively map the interactome of 28 out of 29 ARF and ARL proteins in two cellular models. Through this approach, we identified ~3000 high-confidence proximal interactors, enabling us to assign subcellular localizations to the family members. Notably, we uncovered previously undefined localizations for ARL4D and ARL10. Clustering analyses further exposed the distinctiveness of the interactors identified with these two GTPases. We also reveal that the expression of the understudied member ARL14 is confined to the stomach and intestines. We identified phospholipase D1 (PLD1) and the ESCPE-1 complex, more precisely SNX1, as proximity interactors. Functional assays demonstrated that ARL14 can activate PLD1 in cellulo and is involved in cargo trafficking via the ESCPE-1 complex. Overall, the BioID data generated in this study provide a valuable resource for dissecting the complexities of ARF and ARL spatial organization and signaling.

Regulatory constraints in the evolution of the tetrapod limb anterior-posterior polarity.

The anterior to posterior (A-P) polarity of the tetrapod limb is determined by the confined expression of Sonic hedgehog (Shh) at the posterior margin of developing early limb buds, under the control of HOX proteins encoded by gene members of both the HoxA and HoxD clusters. Here, we use a set of partial deletions to show that only the last four Hox paralogy groups can elicit this response: that is, precisely those genes whose expression is excluded from most anterior limb bud cells owing to their collinear transcriptional activation. We propose that the limb A-P polarity is produced as a collateral effect of Hox gene collinearity, a process highly constrained by its crucial importance during trunk development. In this view, the co-option of the trunk collinear mechanism, along with the emergence of limbs, imposed an A-P polarity to these structures as the most parsimonious solution. This in turn further contributed to stabilize the architecture and operational mode of this genetic system.

Netrin1 and reelin signaling are required for the migration of anterolateral system neurons in the embryonic spinal cord.

ABSTRACT: Projection neurons of the spinal cord dorsal horn which transmit pain, itch, and temperature information to the brain comprise the anterolateral system (AS). A recent molecular and genetic study showed that many developing AS neurons express the transcription factor Phox2a and provided insights into the mechanisms of their ontogeny and wiring of nociceptive neuronal circuits. Here, we show that the loss of the axonal guidance and neuronal migration signal netrin1 results in impaired migration of mouse Phox2a+ AS neurons into the spinal lamina I. Furthermore, we show that in the absence of Dab1, an intracellular transducer of the neuronal migration signal reelin, the migration of spinal lamina V and lateral spinal nucleus Phox2a+ AS neurons is impaired, in line with deficits in nociception seen in mice with a loss of reelin signaling. Together, these results provide evidence that netrin1 and reelin control the development of spinal nociceptive projection neurons, suggesting a mechanistic explanation for studies that link sequence variations in human genes encoding these neurodevelopmental signals and abnormal pain sensation.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration.

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1-/- mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

Angiopoietin-like 2 is essential to aortic valve development in mice.

Aortic valve (AoV) abnormalities during embryogenesis are a major risk for the development of aortic valve stenosis (AVS) and cardiac events later in life. Here, we identify an unexpected role for Angiopoietin-like 2 (ANGPTL2), a pro-inflammatory protein secreted by senescent cells, in valvulogenesis. At late embryonic stage, mice knocked-down for Angptl2 (Angptl2-KD) exhibit a premature thickening of AoV leaflets associated with a dysregulation of the fine balance between cell apoptosis, senescence and proliferation during AoV remodeling and a decrease in the crucial Notch signalling. These structural and molecular abnormalities lead toward spontaneous AVS with elevated trans-aortic gradient in adult mice of both sexes. Consistently, ANGPTL2 expression is detected in human fetal semilunar valves and associated with pathways involved in cell cycle and senescence. Altogether, these findings suggest that Angptl2 is essential for valvulogenesis, and identify Angptl2-KD mice as an animal model to study spontaneous AVS, a disease with unmet medical need.

Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function.

Vertebrate HoxA and HoxD cluster genes are required for proper limb development. However, early lethality, compensation and redundancy have made a full assessment of their function difficult. Here we describe mice that are lacking all Hoxa and Hoxd functions in their forelimbs. We show that such limbs are arrested early in their developmental patterning and display severe truncations of distal elements, partly owing to the absence of Sonic hedgehog expression. These results indicate that the evolutionary recruitment of Hox gene function into growing appendages might have been crucial in implementing hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages. Accordingly, these mutant limbs may be reminiscent of an ancestral trunk extension, related to that proposed for arthropods.

Recombination between inverted loxP sites is cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.

The loxP/Cre recombination system is a widely used tool for mouse functional genomics, in particular for in vivo conditional mutagenesis. Depending on the relative orientation and position of loxP sites, Cre-mediated recombination can result in a variety of targeted genomic rearrangements. It was previously reported that loss of the loxP-carrying chromosome can occur when loxP sites are arranged in inverse orientation. By using a chromosome 2 carrying inverted loxP sites, we found that Cre-mediated recombination not only causes chromosomal loss but also triggers apoptosis. We show that targeted recombination between inverted loxP sites (TRIP) triggers cell death specifically in proliferating Cre-expressing cells, and we provide evidence that TRIP is an efficient tool to ablate proliferating cells within genetically defined cell populations. Furthermore, the procedure requires only a simple, one-step intercross but neither the use of toxins nor the additional step of prodrug injection. With the large repertoire of tissue-specific or inducible Cre-expressing transgenes available, TRIP-mediated cell ablation is valuable to investigate the function of a large variety of cell populations in the context of a whole organism, which includes mechanisms underlying organ development and tissue homeostasis.

Transcriptional Trajectories in Mouse Limb Buds Reveal the Transition from Anterior-Posterior to Proximal-Distal Patterning at Early Limb Bud Stage.

Limb patterning relies in large part on the function of the Hox family of developmental genes. While the differential expression of Hox genes shifts from the anterior-posterior (A-P) to the proximal-distal (P-D) axis around embryonic day 11 (E11), whether this shift coincides with a more global change of A-P to P-D patterning program remains unclear. By performing and analyzing the transcriptome of the developing limb bud from E10.5 to E12.5, at single-cell resolution, we have uncovered transcriptional trajectories that revealed a general switch from A-P to P-D genetic program between E10.5 and E11.5. Interestingly, all the transcriptional trajectories at E10.5 end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P-D identity. Moreover, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. Among these are Hoxa13 and Hoxd13 (Hox13 hereafter), which start to be expressed around E10.5, and importantly the binding of the HOX13 factors was observed within or in the neighborhood of several of the distal limb genes. Our data are consistent with previous evidence suggesting that the transition from the early/proximal to the late/distal transcriptome of the limb mesenchyme largely relies on HOX13 function. Based on these results and the evidence that HOX13 factors restrict Hoxa11 expression to the proximal limb, in progenitor cells of the zeugopod, we propose that HOX13 act as a key determinant of P-D patterning.

HOX13-dependent chromatin accessibility underlies the transition towards the digit development program.

Hox genes encode transcription factors (TFs) that establish morphological diversity in the developing embryo. The similar DNA-binding motifs of the various HOX TFs contrast with the wide-range of HOX-dependent genetic programs. The influence of the chromatin context on HOX binding specificity remains elusive. Here, we used the developing limb as a model system to compare the binding specificity of HOXA13 and HOXD13 (HOX13 hereafter), which are required for digit formation, and HOXA11, involved in forearm/leg development. We find that upon ectopic expression in distal limb buds, HOXA11 binds sites normally HOX13-specific. Importantly, these sites are loci whose chromatin accessibility relies on HOX13. Moreover, we show that chromatin accessibility specific to the distal limb requires HOX13 function. Based on these results, we propose that HOX13 TFs pioneer the distal limb-specific chromatin accessibility landscape for the proper implementation of the distal limb developmental program.