Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape.

The hindlimb and external genitalia of present-day tetrapods are thought to derive from an ancestral common primordium that evolved to generate a wide diversity of structures adapted for efficient locomotion and mating in the ecological niche occupied by the species. We show that despite long evolutionary distance from the ancestral condition, the early primordium of the mouse external genitalia preserved the capacity to take hindlimb fates. In the absence of Tgfbr1, the pericloacal mesoderm generates an extra pair of hindlimbs at the expense of the external genitalia. It has been shown that the hindlimb and the genital primordia share many of their key regulatory factors. Tgfbr1 controls the response to those factors by modulating the accessibility status of regulatory elements that control the gene regulatory networks leading to the formation of genital or hindlimb structures. Our work uncovers a remarkable tissue plasticity with potential implications in the evolution of the hindlimb/genital area of tetrapods, and identifies an additional mechanism for Tgfbr1 activity that might also contribute to the control of other physiological or pathological processes.

Tgfbr1 regulates lateral plate mesoderm and endoderm reorganization during the trunk to tail transition.

During the trunk to tail transition the mammalian embryo builds the outlets for the intestinal and urogenital tracts, lays down the primordia for the hindlimb and external genitalia, and switches from the epiblast/primitive streak to the tailbud as the driver of axial extension. Genetic and molecular data indicate that Tgfbr1 is a key regulator of the trunk to tail transition. Tgfbr1 has been shown to control the switch of the neuro mesodermal-competent cells from the epiblast to the chordo-neural hinge to generate the tail bud. We now show that Tgfbr1 signaling also controls the remodeling of the lateral plate mesoderm (LPM) and of the embryonic endoderm associated with the trunk to tail transition. In the absence of Tgfbr1 the two LPM layers do not converge at the end of the trunk, extending instead as separate layers enclosing the celomic cavity until the caudal embryonic extremity, and failing to activate markers of primordia for the hindlimb and external genitalia. However, this extended LPM, does not exhibit the molecular signatures characteristic of this tissue in the trunk. The vascular remodeling involving the dorsal aorta and the umbilical artery leading to the connection between embryonic and extraembryonic circulation was also affected in the Tgfbr1 mutant embryos. Similar alterations in the LPM and vascular system were also observed in Isl1 null mutants, indicating that this factor acts in the regulatory cascade downstream of Tgfbr1 in LPM-derived tissues. In addition, in the absence of Tgfbr1 the embryonic endoderm fails to expand to form the endodermal cloaca and to extend posteriorly to generate the tail gut. We present evidence suggesting that the remodeling activity of Tgfbr1 in the LPM and endoderm results from the control of the posterior primitive streak fate after its regression during the trunk to tail transition. Our data, together with previously reported observations, place Tgfbr1 at the top of the regulatory processes controlling the trunk to tail transition.

Regulatory changes associated with the head to trunk developmental transition.

BACKGROUND: Development of vertebrate embryos is characterized by early formation of the anterior tissues followed by the sequential extension of the axis at their posterior end to build the trunk and tail structures, first by the activity of the primitive streak and then of the tail bud. Embryological, molecular and genetic data indicate that head and trunk development are significantly different, suggesting that the transition into the trunk formation stage involves major changes in regulatory gene networks. RESULTS: We explored those regulatory changes by generating differential interaction networks and chromatin accessibility profiles from the posterior epiblast region of mouse embryos at embryonic day (E)7.5 and E8.5. We observed changes in various cell processes, including several signaling pathways, ubiquitination machinery, ion dynamics and metabolic processes involving lipids that could contribute to the functional switch in the progenitor region of the embryo. We further explored the functional impact of changes observed in Wnt signaling associated processes, revealing a switch in the functional relevance of Wnt molecule palmitoleoylation, essential during gastrulation but becoming differentially required for the control of axial extension and progenitor differentiation processes during trunk formation. We also found substantial changes in chromatin accessibility at the two developmental stages, mostly mapping to intergenic regions and presenting differential footprinting profiles to several key transcription factors, indicating a significant switch in the regulatory elements controlling head or trunk development. Those chromatin changes are largely independent of retinoic acid, despite the key role of this factor in the transition to trunk development. We also tested the functional relevance of potential enhancers identified in the accessibility assays that reproduced the expression profiles of genes involved in the transition. Deletion of these regions by genome editing had limited effect on the expression of those genes, suggesting the existence of redundant enhancers that guarantee robust expression patterns. CONCLUSIONS: This work provides a global view of the regulatory changes controlling the switch into the axial extension phase of vertebrate embryonic development. It also revealed mechanisms by which the cellular context influences the activity of regulatory factors, channeling them to implement one of several possible biological outputs.

Shaping Hox gene activity to generate morphological diversity across vertebrate phylogeny.

The importance of Hox genes for the development and evolution of the vertebrate axial skeleton and paired appendages has been recognized for already several decades. The steady growth of genomic sequence data from an increasing number of vertebrate species, together with the improvement of methods to analyze genomic structure and interactions, as well as to control gene activity in various species has refined our understanding of Hox gene activity in development and evolution. Here, I will review recent data addressing the influence of Hox regulatory processes in the evolution of the fins and the emergence of the tetrapod limb. In addition, I will discuss the involvement of posterior Hox genes in the control of vertebrate axial extension, focusing on an apparently divergent activity that Hox13 paralog group genes have on the regulation of tail bud development in mouse and zebrafish embryos.

Characterization of the spectrum of trivalent VAV1-mutation-driven tumours using a gene-edited mouse model.

Mutations in the VAV1 guanine nucleotide exchange factor 1 have been recently found in peripheral T cell lymphoma and nonsmall-cell lung cancer (NSCLC). To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a VAV1 mutant protein that recapitulates the signalling alterations present in the VAV1 mutant subclass most frequently found in tumours. We could not detect any overt tumourigenic process in those mice. However, the concurrent elimination of the Trp53 tumour suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type VAV1 plays in follicular helper T cells. We also found that, in combination with the Kras oncogene, the VAV1 mutant version favours progression of NSCLC. These data indicate that VAV1 mutations play critical, although highly cell-type-specific, roles in tumourigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumours involved.

Assessing Myf5 and Lbx1 contribution to carapace development by reproducing their turtle-specific signatures in mouse embryos.

BACKGROUND: The turtle carapace is an evolutionary novelty resulting from changes in the processes that build ribs and their associated muscles in most tetrapod species. Turtle embryos have several unique features that might play a role in this process, including the carapacial ridge, a Myf5 gene with shorter coding region that generates an alternative splice variant lacking exon 2, and unusual expression patterns of Lbx1 and HGF. RESULTS: We investigated these turtle-specific expression differences using genetic approaches in mouse embryos. At mid-gestation, mouse embryos producing Myf5 transcripts lacking exon 2 replicated some early properties of turtle somites, but still developed into viable and fertile mice. Extending Lbx1 expression into the hypaxial dermomyotomal lip of trunk somites to mimic the turtle Lbx1 expression pattern, produced fusions in the distal part of the ribs. CONCLUSIONS: Turtle-like Myf5 activity might generate a plastic state in developing trunk somites under which they can either enter carapace morphogenetic routes, possibly triggered by signals from the carapacial ridge, or still engage in the development of a standard tetrapod ribcage in the absence of those signals. In addition, trunk Lbx1 expression might play a later role in the formation of the lateral border of the carapace.

The X-linked splicing regulator MBNL3 has been co-opted to restrict placental growth in eutherians.

Understanding the regulatory interactions that control gene expression during the development of novel tissues is a key goal of evolutionary developmental biology. Here, we show that Mbnl3 has undergone a striking process of evolutionary specialization in eutherian mammals resulting in the emergence of a novel placental function for the gene. Mbnl3 belongs to a family of RNA-binding proteins whose members regulate multiple aspects of RNA metabolism. We find that, in eutherians, while both Mbnl3 and its paralog Mbnl2 are strongly expressed in placenta, Mbnl3 expression has been lost from nonplacental tissues in association with the evolution of a novel promoter. Moreover, Mbnl3 has undergone accelerated protein sequence evolution leading to changes in its RNA-binding specificities and cellular localization. While Mbnl2 and Mbnl3 share partially redundant roles in regulating alternative splicing, polyadenylation site usage and, in turn, placenta maturation, Mbnl3 has also acquired novel biological functions. Specifically, Mbnl3 knockout (M3KO) alone results in increased placental growth associated with higher Myc expression. Furthermore, Mbnl3 loss increases fetal resource allocation during limiting conditions, suggesting that location of Mbnl3 on the X chromosome has led to its role in limiting placental growth, favoring the maternal side of the parental genetic conflict.

Epha1 is a cell-surface marker for the neuromesodermal competent population.

The vertebrate body is built during embryonic development by the sequential addition of new tissue as the embryo grows at its caudal end. During this process, progenitor cells within the neuromesodermal competent (NMC) region generate the postcranial neural tube and paraxial mesoderm. Here, we have applied a genetic strategy to recover the NMC cell population from mouse embryonic tissues and have searched their transcriptome for cell-surface markers that would give access to these cells without previous genetic modifications. We found that Epha1 expression is restricted to the axial progenitor-containing areas of the mouse embryo. Epha1-positive cells isolated from the mouse tailbud generate neural and mesodermal derivatives when cultured in vitro. This observation, together with their enrichment in the Sox2+/Tbxt+ molecular phenotype, indicates a direct association between Epha1 and the NMC population. Additional analyses suggest that tailbud cells expressing low Epha1 levels might also contain notochord progenitors, and that high Epha1 expression might be associated with progenitors entering paraxial mesoderm differentiation. Epha1 could thus be a valuable cell-surface marker for labeling and recovering physiologically active axial progenitors from embryonic tissues.

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Isolated Incudostapedial Cholesteatomas: Unique Radiologic and Surgical Features.

OBJECTIVES: Congenital cholesteatomas originate from epithelial tissue present within the middle ear in patients with an intact tympanic membrane, no history of otologic surgery, otorrhea, or tympanic membrane perforation. They are diagnosed by a pearl-like lesion on otoscopy and computed tomography (CT) scan showing an expansile soft-tissue mass. We describe a series of patients with no prior otologic history presenting with progressive unilateral conductive hearing loss and normal otoscopy. The CT scans showed ossicular erosion without obvious soft-tissue mass. Surgery confirmed incudostapedial erosion found to be cholesteatoma. In this study, we characterize the clinical course of patients diagnosed with isolated incudostapedial cholesteatoma (IIC) and review possible pathologic mechanisms. METHODS: Retrospective review of IIC cases treated by the Department of Pediatric Otolaryngology, Rady Children's Hospital, San Diego, 2014 to 2020. Data included patient demographics, clinical features, imaging, surgical findings, and audiologic data. RESULTS: Five patients were diagnosed with IIC (3 [60%] female; mean age at presentation 10.7 years [range 5.5-16.0]). All patients presented with postlingual unilateral conductive hearing loss and normal otoscopy without any past otologic history; delay in diagnosis ranged from 4 months to several years. The CT scans showed ossicular chain erosion with an absent long process of the incus and/or stapes superstructure. All patients underwent middle ear exploration, revealing a thin layer of cholesteatoma in the incudostapedial region, confirmed by histopathology. Mean preoperative speech reception threshold was 55 dB and improved to a mean of 31 dB in the 4 patients who underwent ossicular chain reconstruction. CONCLUSION: Isolated incudostapedial cholesteatoma should be included as a possible etiology in pediatric patients with insidious onset of unilateral conductive hearing loss with normal otoscopy, unremarkable otologic history, and a CT scan showing ossicular abnormality/disruption without notable middle ear mass. These patients should be counseled preoperatively regarding the possibility of cholesteatoma and should undergo middle ear exploration with possible ossiculoplasty.

A Tgfbr1/Snai1-dependent developmental module at the core of vertebrate axial elongation.

Formation of the vertebrate postcranial body axis follows two sequential but distinct phases. The first phase generates pre-sacral structures (the so-called primary body) through the activity of the primitive streak on axial progenitors within the epiblast. The embryo then switches to generate the secondary body (post-sacral structures), which depends on axial progenitors in the tail bud. Here we show that the mammalian tail bud is generated through an independent functional developmental module, concurrent but functionally different from that generating the primary body. This module is triggered by convergent Tgfbr1 and Snai1 activities that promote an incomplete epithelial to mesenchymal transition on a subset of epiblast axial progenitors. This EMT is functionally different from that coordinated by the primitive streak, as it does not lead to mesodermal differentiation but brings axial progenitors into a transitory state, keeping their progenitor activity to drive further axial body extension.

The vertebrate tail: a gene playground for evolution.

The tail of all vertebrates, regardless of size and anatomical detail, derive from a post-anal extension of the embryo known as the tail bud. Formation, growth and differentiation of this structure are closely associated with the activity of a group of cells that derive from the axial progenitors that build the spinal cord and the muscle-skeletal case of the trunk. Gdf11 activity switches the development of these progenitors from a trunk to a tail bud mode by changing the regulatory network that controls their growth and differentiation potential. Recent work in the mouse indicates that the tail bud regulatory network relies on the interconnected activities of the Lin28/let-7 axis and the Hox13 genes. As this network is likely to be conserved in other mammals, it is possible that the final length and anatomical composition of the adult tail result from the balance between the progenitor-promoting and -repressing activities provided by those genes. This balance might also determine the functional characteristics of the adult tail. Particularly relevant is its regeneration potential, intimately linked to the spinal cord. In mammals, known for their complete inability to regenerate the tail, the spinal cord is removed from the embryonic tail at late stages of development through a Hox13-dependent mechanism. In contrast, the tail of salamanders and lizards keep a functional spinal cord that actively guides the tail's regeneration process. I will argue that the distinct molecular networks controlling tail bud development provided a collection of readily accessible gene networks that were co-opted and combined during evolution either to end the active life of those progenitors or to make them generate the wide diversity of tail shapes and sizes observed among vertebrates.

Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse.

Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. •Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing.•Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

Tail Bud Progenitor Activity Relies on a Network Comprising Gdf11, Lin28, and Hox13 Genes.

During the trunk-to-tail transition, axial progenitors relocate from the epiblast to the tail bud. Here, we show that this process entails a major regulatory switch, bringing tail bud progenitors under Gdf11 signaling control. Gdf11 mutant embryos have an increased number of such progenitors that favor neural differentiation routes, resulting in a dramatic expansion of the neural tube. Moreover, inhibition of Gdf11 signaling recovers the proliferation ability of these progenitors when cultured in vitro. Tail bud progenitor growth is independent of Oct4, relying instead on Lin28 activity. Gdf11 signaling eventually activates Hox genes of paralog group 13, which halt expansion of these progenitors, at least in part, by down-regulating Lin28 genes. Our results uncover a genetic network involving Gdf11, Lin28, and Hox13 genes controlling axial progenitor activity in the tail bud.

Deconstructing the molecular mechanisms shaping the vertebrate body plan.

The large display of body shapes and sizes observed among vertebrates ultimately represent variations of a common basic body plan. This likely results from the use of homologous developmental schemes, just differentially tinkered both in amplitude and timing by natural selection. In this review, we will revisit, discuss and combine old ideas with new concepts to update our view on how the vertebrate body is built. Recent advances, particularly at the molecular level, will guide our deconstruction of the individual developmental modules that sequentially produce head, neck, trunk and tail structures, and the transitions between them.

Reassessing the Role of Hox Genes during Vertebrate Development and Evolution.

Since their discovery Hox genes have been at the core of the established models explaining the development and evolution of the vertebrate body plan as well as its paired appendages. Recent work brought new light to their role in the patterning processes along the main body axis. These studies show that Hox genes do not control the basic layout of the vertebrate body plan but carry out region-specific patterning instructions loaded on the derivatives of axial progenitors by Hox-independent processes. Furthermore, the finding that Hox clusters are embedded in functional chromatin domains, which critically impacts their expression, has significantly altered our understanding of the mechanisms of Hox gene regulation. This new conceptual framework has broadened our understanding of both limb development and the evolution of vertebrate paired appendages.

Regulatory landscape of the Hox transcriptome.

Precise regulation of Hox gene activity is essential to achieve proper control of animal embryonic development and to avoid generation of a variety of malignancies. This is a multilayered process, including complex polycistronic transcription, RNA processing, microRNA repression, long noncoding RNA regulation and sequence-specific translational control, acting together to achieve robust quantitative and qualitative Hox protein output. For many such mechanisms, the Hox cluster gene network has turned out to serve as a paradigmatic model for their study. In this review, we discuss current knowledge of how the different layers of post-transcriptional regulation and the production of a variety of noncoding RNA species control Hox output, and how this shapes formation of developmental systems that are reproducibly patterned by complex Hox networks.

A tissue-specific, Gata6-driven transcriptional program instructs remodeling of the mature arterial tree.

Connection of the heart to the systemic circulation is a critical developmental event that requires selective preservation of embryonic vessels (aortic arches). However, why some aortic arches regress while others are incorporated into the mature aortic tree remains unclear. By microdissection and deep sequencing in mouse, we find that neural crest (NC) only differentiates into vascular smooth muscle cells (SMCs) around those aortic arches destined for survival and reorganization, and identify the transcription factor Gata6 as a crucial regulator of this process. Gata6 is expressed in SMCs and its target genes activation control SMC differentiation. Furthermore, Gata6 is sufficient to promote SMCs differentiation in vivo, and drive preservation of aortic arches that ought to regress. These findings identify Gata6-directed differentiation of NC to SMCs as an essential mechanism that specifies the aortic tree, and provide a new framework for how mutations in GATA6 lead to congenital heart disorders in humans.

Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan.

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.