Preliminary observations on isotretinoin-induced ear malformations and pattern formation of the external ear.

Retinoic acid is a morphogenic substance capable of inducing a variety of limb malformations, including duplications and reduction-type defects. Whether retinoic acid plays a similar role in controlling pattern formation of other vertebrate structures is unclear. Many fetuses and infants exposed to isotretinoin (13-cis-retinoic acid) in utero have a characteristic pattern of anomalies, chiefly involving brain, craniofacial, and thymic morphogenesis. Among the craniofacial anomalies, external ear malformations are common and the specific types of auricular malformations include partial duplications, and tissue reductions and displacements. These similarities to the types of limb malformations that retinoic acid can induce suggest that retinoic acid may play an important role in controlling pattern formation of facial structures.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.     

A mechanistic model for morphogenesis and regeneration of limbs and imaginal discs.

When an amphibian limb, cockroach leg or Drosophila imaginal disc is subjected to a surgical operation, it is capable of regenerating or duplicating certain parts. Although the structure of the regenerated tissue varies depending on the location and mass of the amputated or transplanted part, it can be predicted from a set of formal rules, called the polar coordinate model [French et al., (1976) Science 193, 969-983; Bryant et al., (1981) Science 212, 993-1002]. In the polar coordinate model, it is assumed (and experimentally proven) that the juxtaposition of normally non-adjacent cells stimulates cell proliferation locally, which implies that the underlying mechanism which gives positional values to each cell, is also responsible for the control of cell growth. Because locally activated proliferation alters the shape and size of the developmental field, the question of how to control the cell growth is the central problem in the regeneration of the limbs and imaginal discs. In this paper, I propose a possible underlying mechanism for the 'polar coordinate rules', and show how this mechanistic model explains the experimental results using computer simulation. The proposed mechanism is an extension of Turing's model (1952). In addition to the reaction-diffusion of the molecules, cell proliferation is taken into consideration. With appropriate initial conditions, the computer simulation shows that a small mass of cells grows up to form a mature limb, and that the mature limb is able to respond to surgical operations as predicted by the polar coordinate model.     

A small region on the X chromosome of Drosophila regulates a key gene that controls sex determination and dosage compensation

In Drosophila, flies with two X chromosomes are females, with one X chromosome, males. We investigated the presence of sex determining factors on the X chromosome by constructing genotypes with one X and various X-chromosomal duplications. We found that female determining factors are not evenly distributed along the X chromosome as had been previously postulated. A distal duplication covering 35% of the X chromosome promotes female differentiation, a much larger proximal duplication of 60% results in male differentiation. The strong feminizing effect of distal duplications originates from a small segment that, when present in two doses, activates Sxl, a key gene for sex determination and dosage compensation. Our results suggest that Sxl can be activated to intermediate levels.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

Travelling in style: the cell biology of pollen

Pollen grains of flowering plants are highly specialized two- to three-cell gametophytes that deliver sperm to the ovule. This function is achieved as a result of a complex developmental programme, including the coordinated events of meiotic divisions, the production of a unique extracellular matrix, the establishment of cytoplasmic domains, and a determinative asymmetric cell division. After maturation, pollen must interact specifically with the receptive female tissues and germinate a highly polarized pollen tube that rapidly grows through the style to the ovule. Thus, pollen is an excellent model system for the study of meiotic events, cellular organization, cell-cell interactions and polar growth in plant biology.     

Limb development in a "nonmodel" vertebrate, the direct-developing frog Eleutherodactylus coqui.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species.     

Anomalies of the forebrain with radial limb defects: Garcia‐Lurie‐Steinfeld syndrome?

BACKGROUND: Severe anomalies of the forebrain together with radial limb anomalies have been reported in Steinfeld syndrome, XK aprosencephaly, and partial monosomy 13q. Steinfeld syndrome is an extremely variable autosomal dominant condition that, in severe cases, is characterized by holoprosencephaly, radial limb defects, and renal and/or cardiac defects. In mild cases there may be only thumb hypoplasia, ocular coloboma, or oral clefts. XK aprosencephaly, also called Garcia-Lurie syndrome (GLS), is a usually sporadic disorder with radial limb defects and aprosencephaly/atelencephaly. Based on two atypical sibships, autosomal recessive inheritance has been suggested. Two patients with variations of monosomy 13q have been described with atelencephaly but, generally, Steinfeld and XK aprosencephaly patients are chromosomally normal. Holoprosencephaly in 13q deletion patients appears to be due to ZIC2 mutations, but ZIC2 has not been previously tested in Steinfeld syndrome or GLS patients. CASES: We report three sporadic cases with clinical features intermediate between Steinfeld and GLS, including severe forebrain malformations and radial limb defects. All had normal karyotypes, and mutations in ZIC2 were absent in the two cases tested. CONCLUSIONS: In our cases and in the literature there is significant clinical overlap between Steinfeld syndrome and GLS. We propose these conditions may not be nosologically or etiologically distinct. The spectrum of severe forebrain anomalies in these conditions is broader than previously thought and may include some neural tube defects. Mild cases are difficult to identify and the full range of expression remains unknown. Autosomal dominant inheritance with incomplete penetrance and frequent new mutations is postulated. Thorough clinical evaluation is recommended for children with severe forebrain and radial limb defects.     

The polar co-ordinate model for pattern regulation in epimorphic fields: A critical appraisal

The polar co-ordinate model for pattern regulation in epimorphic fields, as first proposed by French, Bryant & Bryant (1976), belongs to a general class of models employing two co-ordinates for describing the field value distribution over a monolayer of cells.I first analyse the constraints imposed by the use of polar co-ordinates for biological modelling. These constraints entail: distinction of the actual and derived spaces for the field with their specific and characteristic mapping modes; representation of the field value or a measure of the morphogenetic activity at various positions in the actual space by two polar co-ordinates—the phase or angle and the radius—in the derived space; and attribution of a singular property to the origin in the derived space, which ought to be mapped onto a putative field centre in the actual space.Upon reformulating the polar co-ordinate model while adhering to these constraints, one realizes that the choice of the shortest route between two points in the derived space applies only to the angular, and not to the radial, co-ordinate. Hence the shortest route between two points cannot be conceived as a straight line connecting them.If we assume that cells have certain limits in resolving differences in the field value, the field will virtually fall into territories of distinctive field values, each of which might encompass more than one cell; cells that lie within a single territory should be mutually interchangeable for prospective developmental pathways which may themselves diversify. The central postulate of the model is that disparate field values brought in apposition will induce a field value intercalation through cell proliferation to restore an unbroken sequence of field values for the newly emerging array of territories. This is assumed universally for the angular component of the field value but it need not apply to the radial component.It follows that angular territories meeting at the field centre will produce a central discontinuity of the field value, leading to a perpetual angular intercalation unless some theoretical provision is made. This may be resolved by any ad hoc hypotheses but also by introducing the concept of degeneracy towards the field centre of the angular co-ordinate down to three distinctive values at the innermost level of radial territories. One can then deduce and generalize various empirically recognized behaviours of epimorphic fields. These include the shortest intercalation rule (which may be modified into the more generally applicable, minor-in-major rule), and the distal transformation and so-called complete circle rules (which may now be unified into the more feasible distalization rule proposed by Bryant, French & Bryant, 1981).The models are assessed against various experimental observations mainly for theoretical consistency. A series of experimental tests for various theoretical alternatives are suggested. This work has simply explored the internal logic of the polar co-ordinate model and no suggestion is made, at this stage, to distinguish different families of alternative models for epimorphic systems on experimental grounds.     

Parental Origin of Interstitial Duplications at 15q11.2-q13.3 in Schizophrenia and Neurodevelopmental Disorders

Duplications at 15q11.2-q13.3 overlapping the Prader-Willi/Angelman syndrome (PWS/AS) region have been associated with developmental delay (DD), autism spectrum disorder (ASD) and schizophrenia (SZ). Due to presence of imprinted genes within the region, the parental origin of these duplications may be key to the pathogenicity. Duplications of maternal origin are associated with disease, whereas the pathogenicity of paternal ones is unclear. To clarify the role of maternal and paternal duplications, we conducted the largest and most detailed study to date of parental origin of 15q11.2-q13.3 interstitial duplications in DD, ASD and SZ cohorts. We show, for the first time, that paternal duplications lead to an increased risk of developing DD/ASD/multiple congenital anomalies (MCA), but do not appear to increase risk for SZ. The importance of the epigenetic status of 15q11.2-q13.3 duplications was further underlined by analysis of a number of families, in which the duplication was paternally derived in the mother, who was unaffected, whereas her offspring, who inherited a maternally derived duplication, suffered from psychotic illness. Interestingly, the most consistent clinical characteristics of SZ patients with 15q11.2-q13.3 duplications were learning or developmental problems, found in 76% of carriers. Despite their lower pathogenicity, paternal duplications are less frequent in the general population with a general population prevalence of 0.0033% compared to 0.0069% for maternal duplications. This may be due to lower fecundity of male carriers and differential survival of embryos, something echoed in the findings that both types of duplications are de novo in just over 50% of cases. Isodicentric chromosome 15 (idic15) or interstitial triplications were not observed in SZ patients or in controls. Overall, this study refines the distinct roles of maternal and paternal interstitial duplications at 15q11.2-q13.3, underlining the critical importance of maternally expressed imprinted genes in the contribution of Copy Number Variants (CNVs) at this interval to the incidence of psychotic illness. This work will have tangible benefits for patients with 15q11.2-q13.3 duplications by aiding genetic counseling.     

The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte

The effect of dimethylsulphoxide (DMSO) on the organization of the microtubular system of the mouse oocyte has been examined. Exposure to DMSO causes the immediate appearance of multiple, cold-resistant microtubular asters associated with the foci of pericentriolar material (PCM) normally present in the oocyte. More prolonged exposure to DMSO leads to progressive disassembly of the spindle, and as a result dispersal of the chromosomes and polar PCM foci occurs, and tubulin polymerization becomes confined to PCM-organized asters. Those astral microtubules located between the PCM foci and the cortex of the oocyte appear to be particularly stable, resulting in the development of lengthening radial bundles of microtubules between the PCM and the surface and the progressive movement of the PCM foci towards the centre of the cell. In contrast, after activation of the oocyte the microtubules generated in the presence of DMSO remain located in a cortical mesh. The effects of DMSO do not appear to be fully reversible in most oocytes. We discuss the implications of these results both for the cytoplasmic organization of the oocyte and zygote, and for the attempts at cryopreservation of human oocytes for therapeutic use in infertility programmes.     

A model generating the pattern of cartilage skeletal elements in the embryonic chick limb.

The development of the vertebrate limb involves the production of a specific external form arising from cell division and other growth processes at the cellular level, and the origin within it of specific patterns of tissues arising by cellular differentiation, of which the pattern of cartilages which pre-figure the limb skeleton is the most striking.In this paper we propose a model for the differentiation (or the preceding determination) process that, using only localized cell to cell interactions, can approximate the cartilage pattern in any limb shape. The model requires cells to modify their metabolism irreversibly at critical threshold levels of a diffusible morphogen which may be made or destroyed by these cells. Restrictions inherent in the successful development of a total limb pattern using this system lead to the prediction that the process is confined to a distal band which has no significant interaction with more proximal regions but within this band the characteristic features of the anterior-posterior axis of the limb develop without additional interactions. Cartilage elements are initiated as single “cells” and expand centrifugally to their final size; these elements developing sequentially along the anterior-posterior axis, showing a distinct polarity of size. The model also predicts that equivalent cartilage elements in all vertebrate limbs will be roughly the same relative size at determination, the extensive range of adult structures arising by differential growth and fusion, possibly controlled by global aspects of the model.It must be emphasized that this model only satisfactorily simulates the anterior-posterior patterning of cartilage elements, the disto-proximal pattern being externally imposed.The final cartilage pattern is shown to be a function of (1) the developing shape of the limb, (2) the position of an initiator region that starts the patterning process and (3) the rate of production of the diffusible morphogen. Using parameters selected with as much realism as possible the model gives a good approximation to the pattern of c cartilages found in the normal chick limb; modifying the shape of the limb to that of the talpid³ mutant produces the characteristics features of the cartilage pattern found in that mutant and modifying the rate of morphogen production simulates patterns resembling those found in some ancestral vertebrate fossil forms.     

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.     

Pathogenesis of developmental defects induced in the rat by amniotic sac puncture

The morphogenesis of developmental defects induced by aminotic sac puncture was studied at the gross and microscopic levels. In fetuses recovered from 15 min to 48 h after amniocentesis, a pattern of hemorrhagic lesions, excessive accumulation of interstitial fluid, followed by tissue necrosis and leading ultimately to the reduction of amputation of distal limb segments and morphological changes in the head, was observed. These changes were indicative of venous stasis, hypervolemia and embryonic oxygen deficiency. Intrauterine compression of the fetus and the obstruction of the fetomaternal circulation were considered to be the primary etiological factors in amniocentesis-induced anomalies which included hemorrhagic lesions, limb reductions and amputations, deformities of the head and abdominal regions, generalized edema and postural moulding.     

Stabilization of a terminal inversion duplication of 8p by telomere capture from 18q

Terminal inversion duplications of the short arm of chromosome 8 are one of the more common chromosome rearrangements in humans. We report an infant with multiple congenital anomalies, in whom karyotype analysis showed a terminal inversion duplication of 8p including additional material at the distal end of the derivative chromosome, shown to be of chromosome 18q origin. Terminal inversion duplications of 8p are the result of meiotic recombination between inverted olfactory gene receptor repeats in 8p. This recombination generates a dicentric intermediate that breaks during anaphase, and the broken chromosome end is stabilized by telomere healing or telomere capture. The origin of the telomeric region in the majority of constitutional chromosome deletions studied to date was shown to be from telomere healing; the de novo addition of telomeric repeats. In the proband a cytogenetically detectable piece of chromosome 18q was present on the distal end of the derivative 8, suggesting that this chromosome was stabilized by telomere capture of 18q. FISH analyses of additional cases may yield information as to whether telomere capture or telomere-healing events are the predominant mechanism of chromosome stabilization in terminal inversion duplications of 8p.