Research

Characterization of the cardio-craniofacial mesoderm during vertebrate embryogenesis

For the past few years, our lab has been focusing on the identification of candidate signaling molecules and tissue-specific transcription factors that regulate cardiac and skeletal muscle formation during early vertebrate embryogenesis. Based on previous and ongoing studies in our lab, we propose that the cardiac and craniofacial developmental programs are tightly linked 1-3. Accordingly, insults to any component of this cardio-craniofacial field may lead to both cardiac and craniofacial abnormalities. Below I outline research projects that have been studied in the lab for the last three years.

Lineage plasticity of the cranial paraxial mesoderm:
The developing heart is a specialized muscular vessel that serves as a pump for both the systemic and pulmonary circuits (Figure 1, Panel A). This extremely complicated organ is highly sensitive to genetic perturbations, which are reflected in the numerous congenital heart defects that affect ~1% of all live births. The multiplicity of cardiac progenitor populations in various vertebrate species is an emerging area of intense focus in many laboratories, due to the enormous therapeutic potential of these avenues for treating heart disease. During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e., lateral plate mesoderm and paraxial mesoderm, respectively). We recently employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Utilizing fate mapping studies, gene expression analyses, and manipulations of signaling pathways in the chick embryo, we demonstrated that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further showed that bone morphogenic protein (BMP) signaling affects the specification of mesoderm cells in the head: application of BMP4 to chick embryos, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm, and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo (Figure 1B, and Figure 3) ¹.

Figure 1. Cardio-craniofacial mesoderm specification
A. An image of the adult four-chambered mammalian heart. B. Cranial paraxial mesoderm cells migrate through the branchial arches (pharyngeal arches), where they differentiate into the skeletal muscle lineage. We demonstrated that some of these mesodermal cells can migrate further towards the aortic sac, which connects the branchial arches to the outflow tract. These cells eventually contribute to the myocardium and endocardium of the outflow tract. The gradual shift from a skeletal muscle to a cardiac cell fate is correlated with the spatiotemporal expression of BMP4. Ectopic application of BMP4 both in vitro and in vivo promoted cardiogenesis in the cranial paraxial mesoderm, and blocks the skeletal muscle differentiation program. C. The vertebrate head is an excellent developmental system for the study of both patterning and differentiation programs. During craniofacial development, progenitor cells derived from the cranial paraxial mesoderm fuse together to form a myofiber, which is attached to a specific skeletal element derived from the cranial neural crest in a highly coordinated manner.

Craniofacial muscle patterning:
Craniofacial development requires the orchestrated integration of multiple interactions among progenitor cells derived from both the cranial paraxial mesoderm and the cranial neural crest (CNC, Figure 1C). In the vertebrate head, mesoderm-derived cells fuse together to form a myofiber, which is attached to specific CNC-derived skeletal elements in a highly coordinated manner. Although it has long been suggested that the CNC plays an indirect role in the formation of the head musculature, the precise molecular underpinnings of this exquisitely tuned process, and the significance of the CNC’s contribution to it, are far less clear. In a recent study we analyzed head skeletal muscle patterning and differentiation in vivo, in three mouse models involving genetic perturbations of CNC development, as well as in CNC-ablated chick embryos. Our results demonstrate that although early specification of the skeletal muscle lineage is CNC-independent, CNC cells play an important role at later developmental stages, regulating the expression patterns of myogenic genes, the migration and axial registration of the mesoderm cells, and the subsequent differentiation of myoblasts in the branchial arches. This study supports a model in which CNC cells control craniofacial development and patterning by regulating positional interactions with mesoderm-derived muscle progenitors that together shape the cranial musculoskeletal architecture during vertebrate embryogenesis (Figure 2) ⁴.

Figure 2. A model for the regulation of skeletal muscle formation by cranial neural crest
Our study on craniofacial muscle development in mouse and chick embryonic models has clarified the extent to which the myogenic program is intrinsic or controlled by extrinsic environmental signals. We provide direct evidence that cranial neural crest cells play diverse and critical roles during skeletal muscle formation in vertebrates

The contribution of Islet1-expressing splanchnic mesoderm cells to distinct jaw muscles reveals significant heterogeneity in head muscle development: Heart development takes place in close apposition to the developing head. The term “cardio-craniofacial morphogenetic field” reflects the intimate developmental relationship between the head, face, and heart, which is also reflected in numerous cardiac and craniofacial birth defects (Hutson and Kirby, 2003). Nathan et al, 5 have characterized the nature of the cardio-craniofacial mesoderm in both chick and mouse embryos, using several lineage tracing and gene expression techniques. At both the cellular and molecular levels, the cardio-craniofacial mesoderm can be divided into two compartments, the cranial paraxial mesoderm, and splanchnic mesoderm (SpM), part of which comprises the anterior heart field (AHF). We have found that each of these compartments contributes to the developing heart in a temporally regulated manner. Following linear heart tube stages, we have found that Isl1+/SpM cells contribute to the distal part of the pharyngeal (branchial) mesoderm, as well as to the cardiac outflow tract. Molecular analyses of the head muscles demonstrated distinct molecular and developmental programs for CPM and Isl1+/SpM-derived branchiomeric muscles. Furthermore, we have provided evidence that the Wnt/β-catenin pathway regulates Isl1 and Nkx2.5 gene expression, presumably by fine-tuning boundary formation within the cardio-craniofacial mesoderm.
Figure 3. A model for the contribution of the cranial paraxial mesoderm (CPM), anterior heart field (AHF) and primary/first heart filed (PHF) to the heart and branchial arches. Cells from the CPM and SHF contribute to both the cardiac outflow tract and the myogenic core within the branchial arches.

Deciphering the embryonic origin(s) of satellite cells in the head musculature:
Numerous studies provide a conceptual framework for understanding the relative contributions of cells of distinct mesodermal origins, to cardiac and skeletal muscle tissues. None of these studies, however, attempted to determine the nature of these putative progenitor cells by successively isolating them and characterizing their lineage relationships. Several recent studies have established that satellite cells attached to trunk muscles derive developmentally from the somites (Gros et al., 2005; Kassar-Duchossoy et al., 2005; Relaix et al., 2005; Schienda et al., 2006). At present, however, molecular and cellular investigations of skeletal muscle progenitors in the head are lacking. In particular, little is known about the embryonic origin(s) of satellite cells within the head musculature. In order to identify the origin(s) of craniofacial satellite cells, we will apply various lineage tracing strategies in both avian and mouse models (see below). In the avian system (A), cranial paraxial mesoderm will be labeled using replication-defective viruses or quail-chick transplantations, and the contribution of these cells to the satellite cell population in the head will then be assessed. In the mouse (B) the Cre-Lox genetic system will be employed to perform long-term lineage tracing using Wnt1-Cre (neural crest), Pax3KI-Cre (trunk skeletal muscle and neural crest), MesP1-Cre (cardio-craniofacial mesoderm) and Isl1-Cre (second heart field) mouse lines crossed with the Z/EG or R26R reporters.

embryonic origins

Acknowledgements
Our work is supported by research grants from the Estelle Funk Foundation for Biomedical Research, Ruth & Allen Ziegler, MINERVA, Israel Science Foundation, GIF Young Investigator Award (E.T.), and the Association Francaise Contre Les Myopathies (AFM). Eldad Tzahor is the incumbent of the Gertrude and Philip Nollman Career Development Chair.

References

Tirosh-Finkel et al. Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract. Development 133, 1943-53 (2006).
Tzahor et al. Antagonists of Wnt and BMP signaling promote the formation of vertebrate head muscle. Genes Dev 17, 3087-99 (2003).
Tzahor & Lassar, A.B. Wnt signals from the neural tube block ectopic cardiogenesis. Genes Dev 15, 255-60. (2001).
Rinon et al. Cranial neural crest cells regulate head muscle patterning and differentiation during vertebrate embryogenesis. Development. 134, 3065-75 (2007).
Nathan, et al. The contribution of Islet1-expressing splanchnic mesoderm cells to distinct jaw muscles reveals significant heterogeneity in head muscle development. Development. Under-revision.