Paraxial mesoderm specifies zebrafish primary motoneuron subtype identity

We provide the first analysis of how a segmentally reiterated pattern of neurons is specified along the anteroposterior axis of the vertebrate spinal cord by investigating how zebrafish primary motoneurons are patterned. Two identified primary motoneuron subtypes, MiP and CaP, occupy distinct locations within the ventral neural tube relative to overlying somites, express different genes and innervate different muscle territories. In all vertebrates examined so far, paraxial mesoderm-derived signals specify distinct motoneuron subpopulations in specific anteroposterior regions of the spinal cord. We show that signals from paraxial mesoderm also control the much finer-grained segmental patterning of zebrafish primary motoneurons. We examined primary motoneuron specification in several zebrafish mutants that have distinct effects on paraxial mesoderm development. Our findings suggest that in the absence of signals from paraxial mesoderm, primary motoneurons have a hybrid identity with respect to gene expression, and that under these conditions the CaP axon trajectory may be dominant.     

Loss of late primitive streak mesoderm and interruption of left-right morphogenesis in the Ednrb(s-1Acrg) mutant mouse

This study characterizes defects associated with abnormal mesoderm development in mouse embryos homozygous for the induced Ednrb(s-1Acrg) allele of the piebald deletion complex. The Ednrb(s-1Acrg) deletion results in recessive embryonic lethality and mutant embryos exhibit a truncated posterior body axis. The primitive streak and node become disfigured, consistent with evidence that cell migration is impaired in newly formed mesoderm. Additional defects related to mesoderm development include notochord degeneration, somite malformations, and abnormal vascular development. Arrested heart looping morphogenesis and a randomized direction of embryonic turning indicate that left-right development is also perturbed. The expression of nodal and leftb, Tgf-beta-related genes involved in a left-determinant signaling pathway, is variably lost in the left lateral plate mesoderm. Mutational analysis has demonstrated that Fgf8 and Brachyury (T) are required for normal mesoderm and left-right development and the asymmetric expression of nodal and leftb. Fgf8 expression in nascent mesoderm exiting the primitive streak is dramatically reduced in mutant embryos, and diminished T expression accompanies the progressive loss of paraxial, lateral, and primitive streak mesoderm. In contrast, axial mesoderm persists and T and nodal appear to be appropriately expressed in their specific domains in the node and notochord. We propose that this mutation disrupts a morphogenetic pathway, likely involving FGF signaling, important for the development of streak-derived posterior mesoderm and lateral morphogenesis.     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

Development alters the expression of calcium currents in chick limb motoneurons

Calcium current types expressed in vertebrate spinal motoneurons have not been previously resolved. We have resolved three types in chick limb motoneurons identified by retrograde labeling and report that dramatic changes in their expression take place during development in vivo. T-, N-, and L-type calcium currents were distinguished on the basis of kinetics, voltage dependence, and unique pharmacological sensitivities. Developmental changes were characterized by studying motoneurons isolated from embryos at three stages spanning neuromuscular system development. T currents were dominant at the earliest stage. Motoneurons from embryos 2 days older showed much reduced T currents and much increased N and L currents. We suggest that mature motoneurons will be dominated by N- and L-type calcium currents and that T current may serve developmental roles.     

The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis

In arthropods, annelids and chordates, segmentation of the body axis encompasses both ectodermal and mesodermal derivatives. In vertebrates, trunk mesoderm segments autonomously and induces segmental arrangement of the ectoderm-derived nervous system. In contrast, in the arthropod Drosophila melanogaster, the ectoderm segments autonomously and mesoderm segmentation is at least partially dependent on the ectoderm. While segmentation has been proposed to be a feature of the common ancestor of vertebrates and arthropods, considering vertebrates and Drosophila alone, it is impossible to conclude whether the ancestral primary segmented tissue was the ectoderm or the mesoderm. Furthermore, much of Drosophila segmentation occurs before gastrulation and thus may not accurately represent the mechanisms of segmentation in all arthropods. To better understand the relationship between segmented germ layers in arthropods, we asked whether segmentation is an intrinsic property of the ectoderm and/or the mesoderm in the crustacean Parhyale hawaiensis by ablating either the ectoderm or the mesoderm and then assaying for segmentation in the remaining tissue layer. We found that the ectoderm segments autonomously. However, mesoderm segmentation requires at least a permissive signal from the ectoderm. Although mesodermal stem cells undergo normal rounds of division in the absence of ectoderm, they do not migrate properly in respect to migration direction and distance. In addition, their progeny neither divide nor express the mesoderm segmentation markers Ph-twist and Ph-Even-skipped. As segmentation is ectoderm-dependent in both Parhyale and holometabola insects, we hypothesize that segmentation is primarily a property of the ectoderm in pancrustacea.     

Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 325–340, 1997     

Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity

Despite extensive studies, there are still many unanswered questions regarding the mechanism of hedgehog signaling and the phylogenic conservation of hedgehog function in vertebrates. For example, whether hedgehog signaling in vertebrates requires smoothened is unclear, and the role of hedgehog activity in zebrafish is controversial. We show that inactivation of smoothened by retroviral insertions in zebrafish results in defects that are characteristic of hedgehog deficiencies, including abnormalities in body size, the central nervous system, adaxial mesoderm, cartilage and pectoral fins. We demonstrate that, as in Drosophila, vertebrate smoothened is essential for hedgehog signaling, and functions upstream of protein kinase A. Further analysis of neural tube defects revealed the absence of lateral floor plate and secondary motoneurons, but the presence of medial floor plate and primary motoneurons in smoothened mutant embryos. Blocking maternal hedgehog signaling by cyclopamine eliminates primary motoneurons, but not medial floor plate. Interestingly, even after inhibition of maternal hedgehog activity, the midbrain dopaminergic neurons still form, and looping of the heart does not randomize in the mutants. We also found decreased proliferation and increased apoptosis in the mutants. Taken together, these data demonstrate the conserved role of vertebrate smoothened in the hedgehog signaling pathway, and reveal similarities and differences of hedgehog function between teleosts and amniotes.     

Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.

Peptide growth factors may play a role in patterning of the early embryo, particularly in the induction of mesoderm. We have explored the role of fibroblast growth factor (FGF) in early Xenopus development by expressing a dominant negative mutant form of the FGF receptor. Using a functional assay in frog oocytes, we found that a truncated form of the receptor effectively abolished wild-type receptor function. Explants from embryos expressing this dominant negative mutant failed to induce mesoderm in response to FGF. In whole embryos the mutant receptor caused specific defects in gastrulation and in posterior development, and overexpression of a wild-type receptor could rescue these developmental defects. These results demonstrate that the FGF signaling pathway plays an important role in early embryogenesis, particularly in the formation of the posterior and lateral mesoderm.     

Genetic dissection of nodal function in patterning the mouse embryo

Loss-of-function analysis has shown that the transforming growth factor-like signaling molecule nodal is essential for mouse mesoderm development. However, definitive proof of nodal function in other developmental processes in the mouse embryo has been lacking because the null mutation blocks gastrulation. We describe the generation and analysis of a hypomorphic nodal allele. Mouse embryos heterozygous for the hypomorphic allele and a null allele undergo gastrulation but then display abnormalities that fall into three distinct mutant phenotypic classes, which may result from expression levels falling below critical thresholds in one or more domains of nodal expression. Our analysis of each of these classes provides conclusive evidence for nodal-mediated regulation of several developmental processes in the mouse embryo, beyond its role in mesoderm formation. We find that nodal signaling is required for correct positioning of the anteroposterior axis, normal anterior and midline patterning, and the left-right asymmetric development of the heart, vasculature, lungs and stomach.     

Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF(-/-) embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF(-/-) embryos suffer significant defects in posterior development. Unlike GCNF(+/+) embryos, GCNF(-/-) embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF(-/-) embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF(-/-) embryos is 13 instead of 25 as in the GCNF(+/+) embryos. Interestingly, the tailbud of GCNF(-/-) embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF(-/-) embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.     

A neural degeneration mutation that spares primary neurons in the zebrafish

We describe an embryonic lethal mutation in the zebrafish Brachydanio rerio that specifically affects the viability of most cells in the embryonic central nervous system (CNS). The mutation ned-1 (b39rl) was induced with gamma-irradiation and segregates as a single recessive allele closely linked to its centromere. It produces massive cell death in the CNS but a small set of specific neurons, including Rohon-Beard sensory neurons, large hindbrain interneurons, and primary motoneurons, survive embryogenesis and are functional. Synaptic connections between embryonic motoneurons and muscle cells appear physiologically normal, and the normally observed spontaneous flexions are present. Correlated with the presence of sensory neurons and interneurons, mutant embryos display reflexive movements in response to mechanical stimulation. Together, the surviving neurons, called primary neurons, form a class of cells that are prominent in size and arise early during development. Thus, this mutation may define a function that is differentially required by developmentally distinguishable sets of cells in the embryonic CNS.     

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.     

Taming the tiger of tissue aggregation: how epithelia control structural assembly of underlying cells

A recent paper by Ninomiya and Winklbauer explores the role of the epithelium in restricting the natural capacity of mesenchymal cells to form amorphous aggregates. They propose the epithelium controls mesoderm adhesion, thereby ensuring that embryos and, by extension, adults are not multilayered balls of cells, but are instead elongate with a linearly segmented architecture.     

The POU domain gene, XlPOU 2 is an essential downstream determinant of neural induction

Bone morphogenetic protein 1 (BMP1) is a metalloprotease that ventralises dorsal mesoderm when overexpressed in early Xenopus embryos. Here we show that Xenopus BMP1 blocks the dorsalising activity of chordin, but not noggin or DeltaxBMPR, when coexpressed in the ventral marginal zone and degrades chordin protein in vitro. We also show that a dominant-negative mutation for XBMP1 (dnBMP1) dorsalises ventral mesoderm in vivo, and blocks degradation of chordin by both XBMP1 and Xolloid, a closely related Xenopus metalloprotease, in vitro. dnBMP1 does not dorsalise ventral mesoderm in UV-irradiated embryos, demonstrating that this activity is dependent upon a functional organiser--the natural source of chordin in Xenopus gastrulae. Our results suggest that XBMP1 may regulate the availability of chordin during vertebrate embryogenesis.