Pluripotent Stem Cells as a Model for EmbryonicPatterning: From Signaling Dynamics to SpatialOrganization in a Dish
Idse Heemskerk1 and Aryeh Warmflash1,2*
1Department of Biosciences, Rice University, Houston, Texas2Department of Bioengineering, Rice University, Houston, Texas
Abstract: In vivo studies have identified the signaling pathways and transcription factors involved in patterning the verte-brate embryo, but much remains unknown about how these are organized in space and time to orchestrate embryogenesis.Recently, embryonic stem cells have been established as a platform for studying spatial pattern formation and differentiationdynamics in the early mammalian embryo. The ease of observing and manipulating stem cell systems promises to fill gaps inour understanding of developmental dynamics and identify aspects that are uniquely human. Developmental Dynamics000:000000, 2016. VC 2016 Wiley Periodicals, Inc.
Key words: human embryonic stem cells; morphogen; gastrulation; dynamics; signaling
Submitted 11 March 2016; First Decision 29 June 2016; Accepted 6 July 2016; Published online 0 Month 2013
Despite its importance, relative to other vertebrate classes, muchremains unknown about mammalian development in general,and human development in particular. Knowledge of humandevelopment has medical applications ranging from directedstem cell differentiation for regenerative medicine (Trounson andMcDonald, 2015) to treatment of childhood cancers (Hatten andRoussel, 2011). Moreover, an anthropocentric worldview makesour own development a fascinating subject of fundamentalresearch. Gaps in our knowledge result, to a large extent, fromthe difficulty in studying mammalian development: It is slow andtakes place in utero. Human development poses a particular chal-lenge because ethical considerations prevent experimentation onhuman embryos past the blastocyst stage.
Although mammals may be challenging to study in vivo, thisis balanced by the fact that pluripotent stem cells derived frommammalian embryos can be maintained in culture, enabling invitro study of early development. Embryonic stem cells (ESCs)provide a powerful complement to in vivo studies of develop-ment, as well as a unique model for human development past theblastocyst stage. However, the strengths of the stem cell model gofar beyond the insight it may provide into specifically mammali-an traits. The possibilities that cell culture provides for directobservation and experimental manipulation allow us to address
general questions about embryonic development that are difficultto answer in any in vivo model, and improve our understandingmore broadly.
In the early embryo, pluripotent cells differentiate into ecto-derm, mesoderm, and endoderm, and organize into a trilaminarstructure, a process known as gastrulation. Gastrulation isorchestrated by morphogens thought to form gradients across theembryo and specify the germ layers in a concentration-dependent manner. Much remains uncertain about the dynamicsand shape of these gradients, the way in which they determinecell fate and how they relate to the size and shape of the embryo.It is also unclear whether findings from model systems such asthe mouse can be applied to human development, or, more gener-ally, which aspects of mammalian development are conserved.
In this article, we will review what is known about the dynam-ics of differentiation and morphogen gradient formation in theearly embryo, in particular during gastrulation. Although ourfocus is on understanding mammalian embryogenesis, we alsoreview studies of embryonic patterning in non-mammalian mod-els, as quantitative studies of signaling and patterning dynamicshave not been performed in mammalian systems, and manyaspects are conserved across vertebrates or even bilaterians. Wewill discuss general gaps in our understanding of vertebrate pat-terning as well as those that are specific to mammals or tohumans. After expanding on the challenges of studying develop-ment in vivo, the strengths of the stem cell model, and the rela-tion between stem cells and the embryo, we will discussdifferentiation, spatial patterning, and interspecies differences.
Grant sponsor: National Science Foundation; Grant number: MCB-1553228; Grant sponsor: Cancer Prevention Research Institute ofTexas; Grant number: RR140073.*Correspondence to: Aryeh Warmflash, Department of Biosciences, RiceUniversity, 6100 Main Street, Houston, Texas 77005. E-mail: firstname.lastname@example.org
Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.24432/abstractVC 2016 Wiley Periodicals, Inc.
DEVELOPMENTAL DYNAMICS 00:0000, 2016DOI: 10.1002/DVDY.24432
Our emphasis will be on the importance of dynamics in morpho-gen gradient interpretation; the need for studying endogenousmorphogens to understand pattern formation; and the significantdifferences that exist between model organisms despite the con-servation of core pathways. We will discuss how stem cell modelshave contributed to our understanding of development to date,and examine the opportunities that they provide to complementin vivo studies and answer open questions in developmentalbiology.
Background and Perspective
Difficulties With In Vivo Models
Unraveling the complexity of embryonic development is a daunt-ing task. In vivo, patterning, growth, and morphogenesis of bothembryonic and extraembryonic tissues all happen simultaneouslywith little ability to separate processes in space or time. Imagingof three-dimensional (3-D) embryos and quantitative analysis ofthe resulting data is technically challenging. Moreover, quantita-tive understanding of development would be greatly aided bymaking subtle perturbations, such as changing the shape of a tis-sue or inhibiting and activating signaling in sequence over rela-tively short timescales, but these types of perturbations areimpossible in vivo. Much has been learned from genetic studies,but there is often difficulty in understanding the role of particulargenes due to partially penetrant phenotypes, even among litter-mates. For example, mouse mutants for the TGFb family ligandBMP4 (Winnier et al., 1995) and the Nodal inhibitors Lefty andCerberus (Perea-Gomez et al., 2002) show a wide range of mor-phological phenotypes, and even vary in whether gastrulationtakes place. Although such phenotypic diversity is hard to inter-pret when it concerns the overall development of an embryo, itmay contain additional information about the gene networkwhen intermediate steps such as the abundance of particular cellfates and expression of target genes are quantitatively evaluated(Corson and Siggia, 2012; Raj, et al., 2010).
Mammalian development poses special challenges because ittakes place in utero. The mouse is the mostly widely studiedmammalian model system due to several advantages: relativelylarge litter sizes, short generation times, and straightforwardgenetic manipulation. Nonetheless, technical difficulties stand inthe way of rapid progress, especially in going beyond identifyinggenes and pathways to a quantitative, systems-level understand-ing of differentiation and patterning. In vivo imaging is essen-tially impossible, and live imaging ex vivo is complicated by highsensitivity to phototoxicity (Piliszek et al., 2011). Moreover, keep-ing embryos healthy ex vivo for an extended period of time isextremely challenging. Although major achievements were madein embryo culture nearly 30 years ago, these predated the live-imaging era and were never widely adapted (Chen and Hsu,1982; Hsu, 1979; Tam, 1998). Recently, a technique allowingobservation of embryos as they progress through implantation invitro has been reported for both mouse and human (Bedzhov andZernicka-Goetz, 2014; Deglincerti et al., 2016; Shahbazi et al.,2016), which is a great step forward in the understanding of thisobscure stage of development. However, whether these embryoswill proceed through gastrulation remains unclear, and even ifthey can, in the human case they will not be permitted to do so,so alternative models remain essential.
In addition to experimental challenges, the mouse has limita-tions as a model for human development, as early mammaliandevelopment is diverse (Eakin and Behringer, 2004) and differ-ences between human and mouse are likely significant. Forexample, the development of extraembryonic tissues, and conse-quently the signaling environment of the embryo proper, is quali-tatively different; also, the mouse embryo is cup-shaped whereasthe human embryo is disc-shaped (Behringer et al., 2000;Dobreva et al., 2010; Rossant, 2015).
Stem Cell Culture to Complement In Vivo Studies
The flip side of slow in utero development is the possibility ofmaintaining ESCs in vitro. Moreover, unlike other model systems,in mammals the formation of the embryonic axes and germ layersegregation is independent of maternal transcripts. These featuresenable an alternative approach to the study of development. Fur-thermore, it has recently been shown that axis formation notonly is independent from maternal transcripts, but takes placewithout any maternal cues, which is encouraging for attempts toreproduce this process in vitro (Bedzhov and Zernicka-Goetz,2014; Bedzhov et al., 2015; Morris et al., 2012).
ESCs are pluripotent, meaning they are capable of differentiat-ing to all the cell types of the body. Traditionally, ESCs haveserved as a model for lineage specification but not patterning dueto the spatial disorganization that results from the majority ofESC differentiation methods. However, in recent years they havebeen made to reproduce spatial processes ranging from earlyembryonic patterning (ten Berge et al., 2008; van den Brink et al.,