Aberrant forebrain signaling during early development underlies the generation of holoprosencephaly and coloboma

In this review, we highlight recent literature concerning the signaling mechanisms underlying the development of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close. It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition, other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly

Abnormalities of embryonic patterning are hypothesized to underlie many common congenital malformations in humans including congenital heart defects (CHDs), left-right disturbances (L-R) or laterality, and holoprosencephaly (HPE). Studies in model organisms suggest that Nodal-like factors provide instructions for key aspects of body axis and germ layer patterning; however, the complex genetics of pathogenic gene variant(s) in humans are poorly understood. Here we report our studies of FOXH1, CFC1, and SMAD2 and summarize our mutational analysis of three additional components in the human NODAL-signaling pathway: NODAL, GDF1, and TDGF1. We identify functionally abnormal gene products throughout the pathway that are clearly associated with CHD, laterality, and HPE. Abnormal gene products are most commonly detected in patients within a narrow spectrum of isolated conotruncal heart defects (minimum 5%-10% of subjects), and far less commonly in isolated laterality or HPE patients (approximately 1% for each). The difference in the mutation incidence between these groups is highly significant. We show that apparent gene dosage discrepancies between humans and model organisms can be reconciled by considering a broader combination of sequence variants. Our studies confirm that (1) the genetic vulnerabilities inferred from model organisms with defects in Nodal signaling are indeed analogous to humans; (2) the molecular analysis of an entire signaling pathway is more complete and robust than that of individual genes and presages future studies by whole-genome analysis; and (3) a functional genomics approach is essential to fully appreciate the complex genetic interactions necessary to produce these effects in humans.     

Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly

Holoprosencephaly (HPE), the most common forebrain malformation, is characterized by an incomplete separation of the cerebral hemispheres. Mutations in the homeobox gene SIX3 account for 1.3% of all cases of human HPE. Using zebrafish-based assays, we have now determined that HPE-associated Six3 mutant proteins function as hypomorphs. Haploinsufficiency of Six3 caused by deletion of one allele of Six3 or by replacement of wild-type Six3 with HPE-associated Six3 mutant alleles was sufficient to recapitulate in mouse models most of the phenotypic features of human HPE. We demonstrate that Shh is a direct target of Six3 in the rostral diencephalon ventral midline (RDVM). Reduced amounts of functional Six3 protein fail to activate Shh expression in the mutant RDVM and ultimately lead to HPE. These results identify Six3 as a direct regulator of Shh expression and reveal a crossregulatory loop between Shh and Six3 in the ventral forebrain.     

A dynamic fate map of the forebrain shows how vertebrate eyes form and explains two causes of cyclopia

Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.     

Neural adaptation to tone sequences in the songbird forebrain: patterns, determinants, and relation to the build-up of auditory streaming

Neural responses to tones in the mammalian primary auditory cortex (A1) exhibit adaptation over the course of several seconds. Important questions remain about the taxonomic distribution of multi-second adaptation and its possible roles in hearing. It has been hypothesized that neural adaptation could explain the gradual “build-up” of auditory stream segregation. We investigated the influence of several stimulus-related factors on neural adaptation in the avian homologue of mammalian A1 (field L2) in starlings (Sturnus vulgaris). We presented awake birds with sequences of repeated triplets of two interleaved tones (ABA–ABA–…) in which we varied the frequency separation between the A and B tones (ΔF), the stimulus onset asynchrony (time from tone onset to onset within a triplet), and tone duration. We found that stimulus onset asynchrony generally had larger effects on adaptation compared with ΔF and tone duration over the parameter range tested. Using a simple model, we show how time-dependent changes in neural responses can be transformed into neurometric functions that make testable predictions about the dependence of the build-up of stream segregation on various spectral and temporal stimulus properties.     

A loss-of-function mutation in the CFC domain of TDGF1 is associated with human forebrain defects

TDGF1 (CRIPTO) is an EGF-CFC family member and an obligate co-receptor involved in NODAL signaling, a developmental program implicated in midline, forebrain, and left-right axis development in model organisms. Previous studies of CFC1 (CRYPTIC), another member of the EGF-CFC family, demonstrated that normal function of this protein is required for proper laterality development in humans. Here we identify a mutation in the conserved CFC domain of TDGF1 in a patient with midline anomalies of the forebrain. The mutant protein is inactive in a zebrafish rescue assay, indicating a role for TDGF1 in human midline and forebrain development.     

Major events in the development of the forebrain

The development of the central nervous system can be divided into a number of phases, each of which is characterized by particular developmental disorders. In recent years, much progress has been made in elucidating the mechanisms by which the forebrain develops and in our understanding of major developmental disorders such as holoprosencephaly and neuronal migration disorders. In this general introduction to this symposium the major stages in the development of the forebrain, its regionalization and the genes involved, and some of the developmental disorders derailing cortical development with subsequent damage to the main cortical fiber connections (pyramidal tract and corpus callosum) are discussed.     

Early steps in the development of the forebrain

The tremendous complexity of the adult forebrain makes it a challenging task to elucidate how this structure forms during embryonic development. Nevertheless, we are beginning to understand how a simple epithelial sheet of ectoderm gives rise to the labyrinthine network of cells that constitutes the functional forebrain. Here, we discuss early events in forebrain development--those that lead to the establishment of the anterior neural plate and the regional subdivision of this territory into the different domains of the prospective forebrain.     

Early postembryonic neural development in the zebrafish: a 3-D reconstruction of forebrain proliferation zones shows their relation to prosomeres.

Based on a section-by-section analysis of the morphology (combined silver/Nissl stain) and of the distribution of proliferation zones (immunohistochemical detection of the proliferating cell nuclear antigen) in the zebrafish (Danio rerio) forebrain at 5 days postfertilization, we created a three-dimensional reconstruction of proliferation zones of that developmental stage. The resulting model visualizes the size, number, location and morphology of forebrain proliferation zones. The latter foreshadow closely adult neuroanatomical forebrain entities. Furthermore, the detailed distribution of proliferation zones in the posterior forebrain - but not in the more anterior secondary prosencephalon - supports a segmental prosomeric organization.     

Ultrastructural localization of fibronectin and laminin in human granulation tissue in relation to capillary development

The distribution of fibronectin (FN) and laminin (LM) at developing capillaries during various developmental stages, from capillary sprouts to relatively developed capillaries, was studied by light- and electron-microscopy immunocytochemistry. By light-microscope, FN immunoreactivity was diffusely distributed throughout the stroma of the granulation tissues, while for LM it was preferentially distributed at the perivascular region with the various developmental stages of the immature capillaries. Ultrastructural study revealed that capillary sprouts were closely surrounded by plentiful deposits of immunoreaction with the FN, but only faintly for LM. Relatively developed capillaries with large and tall endothelium were surrounded by plentiful immunoreactive products with both FN and LM, and immunoreactivities in the cisternae of rER of the endothelium and/or the pericytes were also shown. Cytoplasmic interdigitations between the endothelium and the pericyte of developing capillaries were recognized without an immunoreaction for FN and LM. These results mean that the capillary sprouts are associated with a prepatterned FN-rich and LM-poor perivascular matrix, whereas relatively developed capillaries are associated with a FN- and LM-rich perivascular matrix which would have been produced by the capillary endothelium and/or pericytes.     

Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of␣alveolar RMS by influencing the commitment␣and differentiation of the myogenic cell lineage.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .