The basic vertebrate body plan becomes defined with the establishment of the anterior-posterior (A-P), dorsal-ventral (D-V), and left-right (L-R) axes during gastrulation. Efforts to understand these processes at a molecular level have guided a significant fraction of recent developmental biological research, since disturbances in these embryological steps can influence the development of the forebrain [1
], midline structures , as well as the elaboration of L-R sidedness necessary for the asymmetric development of the brain, heart, lungs, gut, and abdominal organs [3
]. While there are clear differences among distinct vertebrate species, these are often variations on a basic theme [5
] that may allow for meaningful extrapolation to human biology.
Nodal is a member of the TGF-β class of secreted signaling molecules that is implicated in the establishment of the A-P axis and generation of mesoderm and definitive endoderm [6
]. It is also involved in a dose-dependent cell-fate specification of midline precursor cells in the anterior primitive streak; these cells will ultimately form the prechordal plate, notochord, and floorplate [7
]. The subsequent anterior migration of these axial mesendoderm cells, in turn, influences the patterning of the developing central nervous system, including the forebrain. Holoprosencephaly (HPE) is considered a “default state” of forebrain development since, in the absence of midline patterning signals from the prechordal plate signaling center, the future forebrain fails to correctly divide into left and right hemispheres and paired subcortical structures [1
]. Furthermore, the axial midline divides the embryo into left-right compartments that can elaborate different molecular properties to affect asymmetric organogenesis [2,3
]. Therefore, Nodal-like factors are crucial for both the development of midline structures and left-right organ specification.
Once the organizer (or node, in the mouse [7
]) develops at the distal tip of the primitive streak, asymmetric signals (including Nodal- and Gdf1-dependent signals) [10
] are subsequently transmitted to the left lateral plate mesoderm, resulting in a cascade of asymmetrically expressed genes such as Nodal
and the downstream pathway effector Pitx2
]. While the asymmetric left-sided expression of Nodal is transient, the continued asymmetric expression of the Pitx2c
isoform is important for the development of the heart and other similarly asymmetrically patterned organs [12
]. Nodal signals induce the expression of Nodal itself through an autoregulatory loop; these signals simultaneously induce Nodal’s antagonists (the Lefty
genes) and, therefore, establish an intricate mechanism to control the effective level of Nodal expression [5
]. Such a tight regulation of Nodal expression is important, since the effective strength or duration of Nodal signals has a direct influence on cellular response [18
Nodal/TGF-β signals are also essential for the specification of the secondary heart field. The secondary heart field provides a source of myocardial precursor cells in the ventral pharyngeal mesoderm [20
] that, in turn, directly contributes to the growth of the cardiac outflow tract or conotruncus [21
]. Therefore, the impaired signaling of the Nodal pathway that has been implicated in cyclopia, craniofacial, cardiovascular, and situs phenotypes in mice [8
] and other model systems can be plausibly extrapolated to similar phenotypes in humans.
While the total absence of murine Nodal is embryonic lethal [26
], heterozygosity for a null allele and a normal allele is entirely compatible with normal development in the mouse strains examined [6
]. Interestingly, a hypomorphic allele of Nodal and a null allele also causes considerable lethality and severe anterior forebrain truncations, while two copies of the same hypomorphic allele allows 50% of the animals to survive to term, but with a spectrum of craniofacial and cardiac defects [18
]. Lowe et al
. described a different hypomorphic allele of Nodal that also manifested a spectrum of axial, forebrain, left-right visceral and conotruncal cardiac malformations, supporting the notion of graded Nodal signaling in the mouse [15
]. Heterozygosity for Nodal loss-of-function mutations also can genetically interact with other genes such as Smad2
], Activin receptor A [32
], or Gdf1
] to generate a similar spectrum of cyclopia and laterality findings, suggesting that compound transheterozygotes might account for some forms of these defects in humans. These results imply that a spectrum of phenotypes can be generated in the mouse when total levels of Nodal activity are between nil and 50%, and that the effective level of cumulative signaling influences which developmental events can proceed normally.
A summary of the core components of the TGF-β signaling pathway utilized by human NODAL [MIM*601265], and the Nodal-like factor GDF1 [MIM*602880], is presented in . Both TGF-β class proteins (see 34–35 for reviews) are structurally similar and interact with a similar array of receptors and downstream effectors. Given that we had previously characterized genetic lesions affecting CFC1
in our patients [36
] we now turned our attention to evaluating the potential role(s) of human NODAL
. Here we describe new NODAL mutations and determine their activity in a new bioassay. We find functionally abnormal mutations in NODAL’s signal sequence, pro domain and mature domain. We also report on two common polymorphisms in NODAL’s pro-domain that affect NODAL activity, and note several cases where the presence of a common weak allele appears to be predictive of disease severity.
The essential components of Nodal signaling