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.

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.

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.

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.