Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.-Jung, K. M., Kim, Y. M., Keyte, A. L., Biegler, M. T., Rengaraj, D., Lee, H. J., Mello, C. V., Velho, T. A. F., Fedrigo, O., Haase, B., Jarvis, E. D., Han, J. Y. Identification and characterization of primordial germ cells in a vocal learning Neoaves species, the zebra finch.

Heterochrony and developmental timing mechanisms: changing ontogenies in evolution.

Heterochrony, or a change in developmental timing, is an important mechanism of evolutionary change. Historically the concept of heterochrony has focused alternatively on changes in size and shape or changes in developmental sequence, but most have focused on the pattern of change. Few studies have examined changes in the mechanisms that embryos use to actually measure time during development. Recently, evolutionary studies focused on changes in distinct timekeeping mechanisms have appeared, and this review examines two such case studies: the evolution of increased segment number in snakes and the extreme rostral to caudal gradient of developmental maturation in marsupials. In both examples, heterochronic modifications of the somite clock have been important drivers of evolutionary change.

Evolutionary and developmental origins of the cardiac neural crest: building a divided outflow tract.

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co-evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome.

Developmental origins of precocial forelimbs in marsupial neonates.

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.

Adaptations of the Marsupial Newborn: Birth as an Extreme Environment.

At birth a mammalian neonate enters an extreme environment compared to the intrauterine environment in which it has grown. This transition may be particularly extreme in marsupials because they are born at an exceedingly altricial state, after an exceptionally short gestation. Their stage of development must be considered embryonic by almost any criteria. Yet at this very early stage of development marsupials must travel to the teat, attach and suckle, and have basic functioning of all major physiological systems. In this article, we review the adaptations of the marsupial neonate for survival at an embryonic state, showing that the neonate exhibits a mosaic of accelerations and delays of various tissues and organs as well as several special adaptations to produce the functioning newborn. We then discuss the development of the craniofacial region, the body axis and limbs in order to detail some of the major changes to development leading to this uniquely configured neonate. We show that marsupial development arises out of a variety of heterochronies (changes in relative timing of events) and heterotopies (changes in location of specific developmental events) at the genetic, cellular and organ level. We argue that these data support hypotheses that many of the specific patterns seen in marsupial development arise from the basic constraint of embryonic energetic and tissue resources. Finally ideas on the evolutionary context of the marsupial developmental strategy are briefly reviewed. Anat Rec, 2019. © 2018 Wiley Periodicals, Inc. Anat Rec, 303:235-249, 2020. © 2018 American Association for Anatomy.

Temperature-activated ion channels in neural crest cells confer maternal fever-associated birth defects.

Birth defects of the heart and face are common, and most have no known genetic cause, suggesting a role for environmental factors. Maternal fever during the first trimester is an environmental risk factor linked to these defects. Neural crest cells are precursor populations essential to the development of both at-risk tissues. We report that two heat-activated transient receptor potential (TRP) ion channels, TRPV1 and TRPV4, were present in neural crest cells during critical windows of heart and face development. TRPV1 antagonists protected against the development of hyperthermia-induced defects in chick embryos. Treatment with chemical agonists of TRPV1 or TRPV4 replicated hyperthermia-induced birth defects in chick and zebrafish embryos. To test whether transient TRPV channel permeability in neural crest cells was sufficient to induce these defects, we engineered iron-binding modifications to TRPV1 and TRPV4 that enabled remote and noninvasive activation of these channels in specific cellular locations and at specific developmental times in chick embryos with radio-frequency electromagnetic fields. Transient stimulation of radio frequency-controlled TRP channels in neural crest cells replicated fever-associated defects in developing chick embryos. Our data provide a previously undescribed mechanism for congenital defects, whereby hyperthermia activates ion channels that negatively affect fetal development.

Opossum (Monodelphis domestica): A Marsupial Development Model.

INTRODUCTIONMonodelphis domestica is the most commonly used laboratory marsupial. In addition to the many factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. In this article, we present an overview of aspects of its biology and its use as a model organism. We also discuss basic care, breeding, embryo manipulation, and modifications of common techniques for the study of the development of this species.

Basic Maintenance and Breeding of the Opossum Monodelphis domestica.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. Monodelphis are easily maintained and bred in the lab. To do extensive embryonic work, a reasonably large breeding colony must be maintained. A colony of ~100 animals (~3:1 female:male ratio) allows for sacrifice of up to 12 pregnant females per month for experimental purposes, as well as for replenishment of the colony. However, because adults will fight and often kill one another if kept in the same cage for prolonged periods, we have developed a special breeding protocol that provides high rates of breeding success (75%-90%), with minimal injury due to fighting. Here, we outline this breeding strategy and describe how to successfully maintain a colony of Monodelphis in a laboratory setting.

Monodelphis whole-embryo culture.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. The embryos of Monodelphis, like those of other marsupials, can be cultured in vitro. The length of embryo viability depends in part on the stage at which culture begins, but embryos of different species of marsupials have been cultured for 18 h to almost 72 h. Good culture results for Monodelphis have been obtained using the method presented here. Embryos can be manipulated and then placed in the incubator. We have applied this technique most commonly to embryos at stages 23-25; they have retained viability and normal development through stage 26 when embryos would begin to implant in vivo.

Harvesting monodelphis embryos.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. Monodelphis embryos are easily harvested, as described in this protocol. Depending on the specific use for the embryo, there may be slight differences in euthanasia procedure, fixation, and embryo treatment. Most commonly, specimens will be used for anatomical or molecular (e.g., in situ hybridization) techniques, in which case they will be fixed in standard fixatives appropriate for the particular protocol.

Whole-mount in situ hybridization in monodelphis embryos.

INTRODUCTIONMonodelphis domestica, the gray, short-tailed, or laboratory opossum, is the most commonly used laboratory marsupial. In addition to the factors that make it a convenient laboratory animal (small size, ease of care, nonseasonal breeding), it is the first marsupial whose genome has been sequenced. Monodelphis has proven useful as a model organism for studies on spinal cord regeneration, ultraviolet (UV)-induced melanoma, and genetic influences on cholesterol, as well as comparative studies of the immune system. In addition, Monodelphis has been used to understand the basic functions of the olfactory system and the role of various olfactory chemicals in social and reproductive behavior. Recently, Monodelphis has been used to understand fundamental aspects of marsupial development, anatomy, evolution, and evolutionary consequences of the derived marsupial mode of development and reproduction. This protocol details whole-mount in situ hybridization of Monodelphis embryos, but it is broadly applicable to any marsupial. Special conditions have been included throughout the protocol for various stages of marsupial embryos. Nevertheless, whole, preterm embryonic stages (~stage 33 to birth) have proven to be difficult to work with because formation of the cuticle prevents probe and antibody penetration.

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.).

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.