Germline Transgenic Methods for Tracking Cells and Testing Gene Function During Regeneration in the Axolotl

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum

Background: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. Results: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. Conclusions: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.Fil: Riquelme Guzmán, Camilo. Technische Universität Dresden; AlemaniaFil: Schuez, Maritta. Technische Universität Dresden; AlemaniaFil: Böhm, Alexander. Technische Universität Dresden; AlemaniaFil: Knapp, Dunja. Technische Universität Dresden; AlemaniaFil: Edwards Jorquera, Sandra. Technische Universität Dresden; AlemaniaFil: Ceccarelli, Alberto Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Chara, Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Argentina de la Empresa; Argentina. Technische Universität Dresden; AlemaniaFil: Rauner, Martina. Universitätsklinikum Carl Gustav Carus; AlemaniaFil: Sandoval Guzmán, Tatiana. Universitätsklinikum Carl Gustav Carus; Alemania. Technische Universität Dresden; Alemani

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development

A conserved regulatory program initiates lateral plate mesoderm emergence across chordates

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo

Neurotrophin Receptor Homolog (NRH1) proteins regulate mesoderm formation and apoptosis during early Xenopus development

Recent experiments suggest that Xenopus Neurotrophin Receptor Homolog 1 (NRH1) proteins act through the planar cell polarity pathway to regulate convergent extension movements during gastrulation and neurulation. We show in this paper that NRH1 proteins are also required for the proper expression of mesodermally expressed genes such as Xbra and Chordin, and to a lesser extent, of Xwnt11. Loss of NRH1 function is followed, during gastrula and neurula stages, by a dramatic increase in apoptosis. Apoptosis is delayed by injection of Xbra RNA, suggesting that cell death is a consequence, at least in part, of the down-regulation of this gene, and it is also delayed by expression of activated forms of Rho, Rac and Cdc42. These small GTPases have previously been implicated in the planar cell polarity pathway in Xenopus and, in other systems, in the regulation of apoptosis. We conclude that the effects of NRH1 proteins include the regulation of mesodermal gene expression and that the disruption of gastrulation that is caused by their loss of function is a consequence of the down-regulation of Xbra and other genes, in addition to direct interference with the planar cell polarity pathway. The apoptosis observed in embryos lacking NRH1 function is not an indirect consequence of the disruption of gastrulation, and indeed it may contribute to the observed morphological defects

Neural crest does not contribute to the neck and shoulder in the axolotl (Ambystoma mexicanum).

BACKGROUND: A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. RESULTS: We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. CONCLUSIONS: Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution

Tissue- and time-directed electroporation of CAS9 protein–gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration

info:eu-repo/semantics/publishe

Results of additional experiments.

<p><b>a</b>, Sagittal section through the neck epaxial muscles in between the scapular tip and occipital region of the skull; this region is devoid of any neural crest-derived connective tissue. Only intersegmental nerves are present along the intermuscular septae are GFP+ (green arrowheads). <b>b–e</b>, transverse sections through the shoulder girdle region of a juvenile (1 month) containing two GFP+ neural folds (see Fig. <b>3a</b>). The framed area in (<b>b</b>) is enlarged in (<b>c–e</b>). <b>c–e</b>, GFP+ spinal nerves close to the shoulder girdle cartilage (<b>c</b>) and Myelin Basic Protein+ cells (anti-MBP-Cy5 immunostaining) in (<b>d</b>) are co-localized (<b>e</b>) as indicated with white arrowheads. <b>f–h,</b> medial aspect of the right shoulder girdle (soft tissues included) of a mature axolotl (3 years) containing two short GFP+ neural fold fragments on either side (same experiment as in (Fig. <b>2a</b>), but with short double- sided graft). <b>f</b>, bright field micrograph of an isolated shoulder girdle whole mount with framed areas enlarged in (<b>g</b>) and (<b>h</b>). The dorsal border of the ossified part of the scapulo-coracoid is indicated with black arrows. <b>g</b>, GFP+ spinal nerves over the GPP-negative ossified scapulo-coracoid. <b>h</b>, nerve net in the muscles connecting to the scapula. GFP+ cells are not present in muscle attachment sites (empty white arrowheads) and the tip of the scapula of somitic origin (white asterisks). Abbreviations: tr2, transverse process of the second vertebra; occ, occipital bone; other abbr. as in Figs. <b>1</b>–<b>3</b>. Scale bars: <b>a–e :</b> 100 µm, <b>f–h :</b> 5 mm.</p