Cranial effects of retinoic acid in the loop-tail (Lp) mutant mouse

The effects of retinoic acid (RA) on the manifestation and nature of neural tube defects (NTD) in heterozygous embryos of mutant mice carrying the gene loop-tail (Lp) and in normal (+/+) littermates and embryos from normal homozygous matings were compared with NTD that occur in untreated abnormal homozygous (Lp/Lp) embryos. A single intraperitoneal dose (5 mg/kg) of RA administered at 9 AM or 3 PM on day 8 of gestation induced NTD in +/+ as well as Lp/+ embryos removed on day 12 of gestation. All of the NTD were confined to the brain and consisted of exencephaly involving the diencephalon, mesencephalon, and metencephalon. In neither phenotype (Lp/+; +/+) was the massive exencephaly and myeloschisis characteristic of untreated Lp/Lp embryos produced; thus, it is possible that the teratogenic mechanisms of RA-induced defects and of Lp-induced defects may differ.     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog

 The hedgehog family of intercellular signalling molecules have essential functions in patterning both Drosophila and vertebrate embryos. Drosophila has a single hedgehog gene, while vertebrates have evolved at least three types of hedgehog genes (the Sonic, Desert and Indian types) by duplication and divergence of a single ancestral gene. Vertebrate Sonic-type genes typically show conserved expression in the notochord and floor plate, while Desert- and Indian-type genes have different patterns of expression in vertebrates from different classes. To determine the ancestral role of hedgehog in vertebrates, I have characterised the hedgehog gene family in amphioxus. Amphioxus is the closest living relative of the vertebrates and develops a similar body plan, including a dorsal neural tube and notochord. A single amphioxus hedgehog gene, AmphiHh, was identified and is probably the only hedgehog family member in amphioxus, showing the duplication of hedgehog genes to be specific to the vertebrate lineage. AmphiHh expression was detected in the notochord and ventral neural tube, tissues that express Sonic-type genes in vertebrates. This shows that amphioxus probably patterns its ventral neural tube using a molecular pathway conserved with vertebrates. AmphiHh was also expressed on the left side of the pharyngeal endoderm, reminiscent of the left-sided expression of Sonic hedgehog in chick embryos which forms part of a pathway controlling left/right asymmetric development. These data show that notochord, floor plate and possibly left/right asymmetric expression are ancestral sites of hedgehog expression in vertebrates and amphioxus. In vertebrates, all these features have been retained by Sonic-type genes. This may have freed Desert-type and Indian-type hedgehog genes from selective constraint, allowing them to diverge and take on new roles in different vertebrate taxa. Received: 20 July 1998 / Accepted: 23 September 1998     

Dual origin of the floor plate in the avian embryo

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen's node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen's node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3beta and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3beta transiently, Shh continuously and other floor-plate characteristic genes such as NETRIN: In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.     

A cell-type-specific abnormality of cell proliferation in mutant (curly tail) mouse embryos developing spinal neural tube defects

The mouse mutant curly tail (ct) provides a model system for studies of neurulation mechanisms. 60% of ct/ct embryos develop spinal neural tube defects (NTD) as a result of delayed neurulation at the posterior neuropore whereas the remaining 40% of embryos develop normally. In order to investigate the role of cell proliferation during mouse neurulation, cell cycle parameters were studied in curly tail embryos developing spinal NTD and in their normally developing litter-mates. Measurements were made of mitotic index, median length of S-phase and percent reduction of labelling index during a [3H]thymidine pulse-chase experiment. These independent measures of cell proliferation rate indicate a reduced rate of proliferation of gut endoderm and notochord cells in the neuropore region of embryos developing spinal NTD compared with normally developing controls. The incidence of cell death and the relative frequency of mitotic spindle orientations does not differ consistently between normal and abnormal embryos. These results suggest a mechanism of spinal NTD pathogenesis in curly tail embryos based on failure of normal cell proliferation in gut endoderm and notochord.     

Homocysteine modifies development of neurulation and dorsal root ganglia in chick embryos

BACKGROUND: The formation of the neural tube (neurulation) involves two mechanisms: primary and secondary neurulation. In chicks, there is also an overlap zone, where both mechanisms work together. Homocysteine (Hcy) may have an important teratogenic role in neural tube defects (NTD) when folic acid levels are considered normal. Recently, Hcy capability to generate NTD and modify neural crest cell migration has been demonstrated in chick embryos. This study was aimed to evaluate the effects of Hcy on neurulation and the development of the dorsal root ganglia (DRG). METHODS: Chick embryos were treated with L-Hcy thiolactone 20 micromol to produce the highest rate of survival with embryos carrying neural tube defect (NTD) in the spine. Embryos at stages (st) 3-10 were treated and harvested at st 18-23. Only externally normal embryos or those carrying spinal NTD embryos were considered. RESULTS: Histological sections of Hcy-treated embryos showed: open spina bifida (39% of embryos), more than one tube forming the spinal cord (26%), disorganized spinal cord (26%), always affecting lumbosacral regions, probably in the overlap zone. Additionally, 32% of embryos had small and continuous DRG, associated with a slimmed roof plate. Three-dimensional reconstruction showed unsegmented DRG until the C8 ganglion level. There was a 75% reduction of C3 DRG cells in treated embryos in comparison to untreated ganglia. CONCLUSION: Hcy teratogenicity in avian embryos affected the neural tube in the overlap zone, secondary neurulation and the cervical DRG.     

HrzicN,a new Zic family gene of ascidians,plays essential roles in the neural tube and notochord development

Two axial structures, a neural tube and a notochord, are key structures in the chordate body plan and in understanding the origin of chordates. To expand our knowledge on mechanisms of development of the neural tube in lower chordates, we have undertaken isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi. HrzicN expression was detected by whole-mount in situ hybridization in all neural tube precursors, all notochord precursors, anterior mesenchyme precursors and a part of the primary muscle precursors. Expression of HrzicN in a- and b-line neural tube precursors was detected from early gastrula stage to the neural plate stage, while expression in other lineages was observed between the 32-cell and the 110-cell stages. HrzicN function was investigated by disturbing translation using a morpholino antisense oligonucleotide. Embryos injected with HrzicN morpholino ('HrzicN knockdown embryos') exhibited failure of neurulation and tail elongation, and developed into larvae without a neural tube and notochord. Analysis of neural marker gene expression in HrzicN knockdown embryos revealed that HrzicN plays critical roles in distinct steps of neural tube formation in the a-line- and A-line precursors. In particular HrzicN is required for early specification of the neural tube fate in A-line precursors. Involvement of HrzicN in the neural tube development was also suggested by an overexpression experiment. However, analysis of mesodermal marker gene expression in HrzicN knockdown embryos revealed unexpected roles of this gene in the development of mesodermal tissues. HrzicN knockdown led to loss of HrBra (Halocynthia roretzi Brachyury) expression in all of the notochord precursors, which may be the cause for notochord deficiency. Hrsna (Halocynthia roretzi snail) expression was also lost from all the notochord and anterior mesenchyme precurosrs. By contrast, expression of Hrsna and the actin gene was unchanged in the primary muscle precursors. These results suggest that HrzicN is responsible for specification of the notochord and anterior mesenchyme. Finally, regulation of HrzicN expression by FGF-like signaling was investigated, which has been shown to be involved in induction of the a- and b-line neural tube, the notochord and the mesenchyme cells in Halocynthia embryos. Using an inhibitor of FGF-like signaling, we showed that HrzicN expression in the a- and b-line neural tube, but not in the A-line lineage and mesodermal lineage, depends on FGF-like signaling. Based on these data, we discussed roles of HrzicN as a key gene in the development of the neural tube and the notochord.     

Depletion of Mab21l1 and Mab21l2 messages in mouse embryo arrests axial turning, and impairs notochord and neural tube differentiation

BACKGROUND: The nematode mab-21 gene specifies sensory ray cell identity and was first isolated because of its mutant sensory ray defects. Vertebrate Mab21 orthologs have since been identified in mammals and amphibians. In this report, we characterized in detail two Mab21 orthologs in mouse, Mab21l1 and Mab21l2. METHODS: We examined the genomic organizations of Mab21 genes and used northern blot and in situ hybridizations to assay their temporal-spatial expression pattern. Their embryonic functions were revealed by specific attenuation of Mab21 messages with antisense oligos in cultured embryos. RESULTS: Mab21l1 and Mab21l2 have very similar protein make-up and gene structures. Both genes were expressed in overlapping domains of actively differentiating embryonic tissues. In addition, Mab21l1 had unique expression in the lens vesicles and genital tubercle whereas Mab21l2 was expressed in the retinal epithelium and umbilical cord. Mab21l1 and Mab21l2 depleted embryos had severe defects in notochord, neural tube, organogenesis, vasculogenesis, and axial turning. CONCLUSIONS: The findings demonstrate that both Mab21 genes are required in developing embryos for embryonic turning, formation of the notochord, neural tube, and other organ tissues.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Ultrastructural analysis of the midaxial extracellular matrix in spinal dysraphism.

Ultrastructural aspects of the extracellular matrix (ECM) in the midaxial region of dysraphic embryos of the loop-tail (Lp) mutant mouse were analyzed by means of electron microscopy. In 17-23 somite embryos, ultrastructural differences in the ECM occurred with respect to the presence of a pair of long trailing basal laminar strands extending continuously from the ventral notochordal cells to the gut in abnormal (Lp/Lp) embryos, in contrast to short, ragged, discontinuous strands in normal (+/+; Lp/+) embryos. The ultrastructural localization and configuration of fibronectin (FN) and laminin (L) associated with these strands, however, were similar in normals and abnormals. In addition, FN occurred over interstitial bodies, fibrils, and sporadically along the basal laminae of the neural tube (or folds), notochord, gut, and vessels, whereas L was largely confined to the basal laminae. The results indicate that although the ultrastructural pattern of FN and L reactivity are similar in normal and abnormal embryos, a disturbance in the manner whereby the notochord detaches from the gut in dysraphic embryos may be of causal significance in the etiology of dysraphism in this mutant.     

The role of the notochord for epaxial myotome formation in the mouse.

The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals.     

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.     

Retinal axon misrouting at the optic chiasm in mice with neural tube closure defects

In a new mouse mutant, circletail (Crc), failure of neural tube closure (embryonic day [E] 8-9) is associated with errors in retinal axon projection at the optic chiasm (E12-18), such that many axons normally projecting contralaterally instead grow to ipsilateral targets. Although the architecture of the chiasmatic region is altered, neurons and glia containing putative cues for axon guidance are present. The aberrant ipsilateral-projecting cells originate from a nonrandom expansion of the wild-type uncrossed retinal region. These axon pathway defects are found in two other mutants with cephalic neural tube defects (NTD), loop-tail (Lp) and Pax3 (splotch; Sp(2H)). Crc is phenotypically similar to Lp, exhibiting an open neural tube from midbrain to tail (craniorachischisis), while splotch has spina bifida with or without a cranial NTD. The retinal axon abnormalities occur only in the presence of NTD and not in homozygous mutants lacking cranial NTD. Thus, failure of neural tube closure is associated with failure of many retinal axons to cross the ventral midline. This study therefore reveals an unexpected connection between closure of the neural tube at the dorsal midline and development of ventral axon tracts. genesis 27:32-47, 2000.     

Relationship between insecticide-induced short and wry neck and cervical defects visible histologically shortly after treatment of chick embryos

In order to clarify the anatomical precursor of short and wry neck, 48-hr chick embryos were injected with 6.25-200 micrograms of the organophosphate (OP) insecticide diazinon and recovered either at 96 hr for histological evaluation or at 19 days for gross observation. Among embryos recovered at 96 hr, all receiving a dose of 25-200 micrograms showed, in serial cross sections, the cervical notochord severely folded in the vertical, horizontal, and diagonal planes and the adjacent neural tube variously folded (often with branching of its canal), deformed by the notochord, rotated, and/or displaced from the midline. Virtually all embryos injected with 6.25 or 12.5 micrograms were fully free of such abnormalities. The coinjection of 2-pyridinealdoxime methochloride (2-PAM, which protects the embryo from certain OP insecticide-induced teratisms) along with 200 micrograms of diazinon markedly reduced the notochord and neural wry neck at 19 days paralleled the 96-hr cervical histology: pronounced in all embryos receiving greater than or equal to 25 micrograms, virtually nonexistent in those receiving 6.25 or 12.5 micrograms. Though more marked at higher doses, wry neck occurred to varying extents at all doses, 6.25-100 micrograms. We conclude that 1) the primary insecticide effect is upon the notochord rather than the neural tube, 2) short neck is a direct consequence of notochord folding, 3) wry neck is apparently not linked with notochord folding, and 4) vertebral fusion is not the consequence solely of muscle paralysis as argued elsewhere. We propose that the notochord folds because diazinon disrupts normal formation of its sheath.     

Immunohistochemical Analysis of the Development of the Floor Plate- and Notochord-Deprived Neural Tube

To analyze the characteristics of neurons and the ectopic fibers that occur in the floor plate-deprived neural tube, the neural tube was separated ipsilaterally from the floor plate and notochord in chick embryos at H-H stage 12. After fixation, operated embryos were labeled with several monoclonal antibodies for detecting cell types and defining the regional characteristics of the neural tube. On the operated side, the basement membrane of the neural tube showed characteristics similar to that of the alar plates. Many neurons had axons that extended outside of the neural tube but which lacked the antigen normally associated with motoneurons. Fibers from the dorsal root ganglia also displayed an atypical distribution within the neural tube. These observations suggest that the neurons in the alar plate can develop independently from the influence(s) of the floor plate and/or notochord and send their axons outside of the neural tube despite the fact that neurons developed in the alar plate do not send axons into the periphery during normal development. It is likely that inhibitory mechanisms, which normally function to restrict axonal growth to within the neural tube, either do not develop or are prevented from functioning in the basal plate lacking environment.     

Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.