Tubulin and Microtubules in the Early Development of the Axolotl and Other Amphibia

SYNOPSIS. Unfertilized eggs of the axolotl, Ambystoma mexicanum,contain a pool of soluble tubulin accumulated during oogenesis.After initiation of cleavage the tubulin pool decreases somewhatand then remains constant through early development. Some propertiesof tubulin alter during development, but at least some of thesechanges are not due to changes in tubulin perse. However, thetubulin in axolotl oocytes, eggs, and embryos differs in someelectrophoretic properties from tubulin in adult axolotl brainand testis. Equivalent differences were observed in Necturusmaculosus tubulins. Heterogeneity of axolotl tubulins was confirmedby peptide mapping: Different patterns of peptides were formedby specific limited proteolysis of soluble tubulin from eggsand testis. The heterogeneity was more marked in the a thanin the ß subunit. Mobilization of soluble tubulininto the mitotic apparatus depends on the functioning of microtubuleorganizing centers after activation of the egg at fertilization.In eggs of the nc mutant axolotl there is a lesion in some stepof activation, one effect of which is that even though the eggscontain an essentially normal pool of tubulin, microtubulesfail to assemble, no mitotic apparatus forms, and embryonicdevelopment does not begin. These eggs can be partially correctedby injection of heterologous microtubule fragments, which elicitthe mobilization of nc tubulin into arrays of microtubules,followed by initiation of cleavage and development to a partialblastula stage. The results of these experiments are discussedin comparison with other reports in the literature about thefunction of microtubule organizing centers during amphibianegg development.     

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.     

The developmental genetics of pigment mutants in the Mexican axolotl

The Mexican axolotl (Ambystoma mexicanum) provides a well-defined set of color genes which are useful for various types of analyses. These include the a (albino), m (melanoid), ax (axanthic), and d (white) genes. In addition, various combinations of these genes and a number of as yet undescribed mutants also exist. Three of these mutants (a, ax, and m) have defects associated with specific neural-crest-derived pigment cell types. The fourth mutant (d) appears to provide an unsuitable environment for the migration and maintenance of pigment cells. In one case (m), detailed information concerning the specific nature of the genetic defect is available. The goal of this article is to demonstrate ways in which the existing information on the axolotl color genes can best be utilized in terms of understanding not only the mutant phenotypes, but basic concepts in the cell and developmental biology of pigmentation as well. Thus, an attempt has been made to sort through the genetic and biochemical data relevant to these mutants in order to stimulate renewed interest in a more detailed pursuit of such studies.     

Experimental studies on a mutant gene (cl) in the Mexican axoloti which affects cell membrane formation in embryos from cl-cl females

The gene cl exerts a maternal effect in the Mexican axolotl resulting in an abnormal cleavage pattern. The early cleavage furrows appear partially depigmented and never continue completely around the egg. Subsequent divisions display a similar pattern which results in the vegetal hemisphere remaining uncleaved; but some portions of the animal hemisphere continue to cleave normally. Gastrulation is very rarely initiated.Several cytological abnormalities including polyploidy, broken chromosomes and fusion of nuclei are observed in mutant embryos. These abnormalities are likely secondary effects resulting from the absence of cell boundaries in the uncleaved portions of the embryo and account for its limited development. Cytochalasin B treatment of normal fertilized eggs produces phenocopies of the most severely affected mutant embryos. This suggests that the cl gene may directly affect the synthesis and/or distribution of a cell surface component which enables daughter cell membranes to be assembled and to adhere to one another.Cells from mutant blastulae were able to differentiate pigmented epidermis and neural tube when grafted to normal recipient blastulae or neurulae. This suggests that the gene is lethal only to cells derived from the vegetal cytoplasm or cortex, but not lethal to cells inheriting animal cytoplasm from $\text{cl}\text{cl}$ females.     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

Mitogen-induced B-cell differentiation in Xenopus laevis

Abstract. Four genes are known to affect pigmentation in the Mexican axolotl. The purpose of this article is to review previous information pertinent to these genes and to reevaluate such information in light of new evidence that demonstrates (in a preliminary way) how pigments, and subsequently phenotypes, are affected by the various pigment genes. Each of the mutant phenotypes – m (melanoid), ax (axanthic), a (albino), and d (white) - is compared to the wild type (D). All of these genes are recessives, all of them affect phenotypic changes during development, and three of the four ( m, a , and d ) also affect specific biochemical (i.e., pigment) changes during development. In the axolotl, color patterns can be directly correlated to the presence(or absence) of a variety of pigments that are normally found in discrete pigment cells. Qualitative and quantitative analyses of the bright-colored pigments (pteridines and flavins in this case) present in axolotl skin demonstrate that these pigments vary significantly among the various phenotypes under consideration. These analyses raise some interesting questions with regard to how each of the pigment genes is believed to act, and numerous possibilities for continued experimentation are suggested.     

Experimental studies on two mutant genes,r and x,in the Mexican axolotl (Ambystoma mexicanum)

Mutant genes r and x, discovered in two unrelated axolotl stocks, are simple recessives determining autonomous cell lethal traits. These traits become recognizable by their characteristic gill and limb patterns which appear in each at about the same period of development. The life spans of the two mutants are approximately the same. Larvae homozygous for both mutants are easily recognized by their smaller size, reduced gill development, and unusually small eyes. None of the three mutant phenotypes (r/r, x/x, rr/xx) is benefited by parabiosis with a normal larva. Transplants of the forelimb area from all three usually were soon invaded by tissues of host origin, resulting in limbs ranging from those almost normal to those reduced to functionless stumps. Those from r/r donors produced the highest percentage of useful limbs. Transplants of the gill-forming area produced gills of the mutant type which, in all cases, regressed. Distention and rupture of gill vessels led to death of some animals. In others the gills became reduced to mere stubs or even disappeared. The failure of replacement of pharyngeal structures of mutant graft origin resulted in the death of all grafted animals from vascular accidents or by prevention of normal feeding or respiration.     

The axolotl mutants

The Mexican axolotl (Ambystoma mexicanum) has enjoyed wide use in experimental embryology for over 100 yr. Its usefulness has been extended into the area of developmental genetics largely due to the contributions of R. Briggs and R. R. Humphrey at Indiana University. To date over 30 mutants have been described, almost all of which affect development. Some of these have been discovered in inbred strains while others have been uncovered in recent Mexican imports. These mutants can be subdivided into several major classes. Maternal effect mutations lead to deficiencies in informational, structural, or metabolic components of the egg essential to early development prior to the time at which the embryo's own genome becomes active. In contrast, the developmental lethals affect later stages in embryogenesis when both morphogenetic and biochemical events are determined exclusively by the genotype of the embryo. Most lead to death at about feeding stage. Some, the cell lethals, are believed to suffer from fundamental metabolic defects affecting all parts of the embryo. Others affect the development of specific organs or tissues. The developmental nonlethals also affect specific systems, but ones that are not essential to survival. Some affect the development and survival of pigment cells and these, along with isozyme variants, are useful as markers in developmental experiments. A number of the mutants have been studied in detail, but others scarcely at all. The purpose of this review is to bring them to the attention of all developmental biologists in the hope that their potential will be even more widely recognized.     

一种新的家蚕体形突变体——短体蚕（Sq）的遗传分析与基因定位

作为重要经济昆虫和鳞翅目昆虫模式的家蚕Bombyx mori,其突变体是生理学、遗传学、功能基因组学等研究的宝贵资源。作者在家蚕资源保存和遗传分析中发现一种新的体形突变体——短体蚕（Squab,Sq）,其特征是：杂合体（Sq /+）成活,蚕体长只有正常型的约4/5,腹中部略肥大,胸部稍狭小;纯合体（Sq / Sq）胚胎期致死。遗传分析结果表明该突变为显性遗传。通过与各染色体标记基因进行连锁分析,发现突变基因Sq在家蚕第14染色体上;通过与同一染色体上的标记基因青熟油蚕基因（oa）、不洁蚕基因（Di）进行三点测验,将Sq定位在家蚕连锁图谱第14连锁群的34.6 cM位点,表示为Sq（14-34.6）。本研究结果为深入研究和利用该突变体奠定了重要基础。     

Growth, Degrowth, and Irreversible Cell Differentiation in Aurelia aurita

Growth patterns of the Scyphomedusa Aurelia aurita from TomalesBay. California, were examined in the field and in the laboratory.Manipulation of growth patterns demonstrated that degrowth andregrowth are not constrained by initial ievelopmental stage.Although initial degrowth of certain tissues is allometric (e.g.,gonads regress in 5 to 8 days; bell diameter decreases morerapidly at first than do the oral arms), thereafter regressionappears identical to, but reversed from normal growth. Regrowthpatterns are normal. Sexual maturation in the sea does not alwaysalter subsequent capacity for degrowth or regrowth to sexualmaturity in the laboratory, because reproductive and somatictissues do not always degenerate after spawning. Gonadal tissuecan be renewed and maintained in a ripe condition in the laboratoryapparently indefinitely. Sexual maturation is a size-dependentphenomenon, not an agespecific developmental event. Spermatogenesis, once initiated, proceeds irrespective of outsideevents. Labeled spermatogonial cells can continue to differentiateto form sperm even though the gonad containing those cells,and the animal itself, show rapid degrowth. The importance ofthis decoupling of developmental events is discussed. The experimentalimportance of animals with flexible life cycles is emphasized.     

Immunofluorescent studies on titin and myosin in developing hearts of normal and cardiac mutant axolotls

Homozygous recessive cardiac mutant gene c in the axolotl, Ambystoma mexicanum, results in a failure of the embryonic heart to initiate beating. Previous studies show that mutant axolotl hearts fail to form sarcomeric myofibrils even though hearts from their normal siblings exhibit organized myofibrils beginning at stage 34–35. In the present study, the proteins titin and myosin are studied using normal (+/+) axolotl embryonic hearts at stages 26–35. Additionally, titin is examined in normal (+/c) and cardiac mutant (c/c) embryonic axolotl hearts using immunofluorescent microscopy at stages 35–42. At tailbud stage-26, the ventromedially migrating sheets of precardiac mesoderm appear as two-cell-layers. Myosin shows periodic staining at the cell peripheries of the presumptive heart cells at this stage, whereas titin is not yet detectable by immunofluorescent microscopy. At preheartbeat stages 32–33, a myocardial tube begins to form around the endocardial tube. In some areas, periodic myosin staining is found to be separated from the titin staining; other areas in the heart at this stage show a co-localization of the two proteins. Both titin and myosin begin to incorporate into myofibrils at stage 35, when normal hearts initiate beating. Additionally, areas with amorphous staining for both proteins are observed at this stage. These observations indicate that titin and myosin accumulate independently at very early premyofibril stages; the two proteins then appear to associate closely just before assembly into myofibrils. Staining for titin in freshly frozen and paraffin-embedded tissues of normal embryonic hearts at stages 35, 39, and 41 reveals an increased organization of the protein into sarcomeres as development progresses. The mutant siblings, however, first show titin staining only limited to the peripheries of yolk platelets. Although substantial quantities of titin accumulate in mutant hearts at later stages of development (39 and 41), it does not become organized into myofibrils as in normal cells at these stages. © 1994 Wiley-Liss, Inc.     

Genetic Control of Early Embryogenesis in the Red Flour Beetle, Tribolium castaneum

SYNOPSIS. The power of genetic analysis possible with the fruitfly, Drosophila melanogaster, has yielded a detailed understandingof pattern formation controlled by homeotic and segmentationgenes in early embryogenesis. We are studying the genetic regulationof embryogenesis in the red flour beetle, Tribolium castaneum.The dynamic process of germ rudiment formation and sequentialsegmentation exhibited by Tribolium provides a context differentthan Drosophila within which to assess the function of homeoticand segmentation gene homologs. Our analyses of the genes inthe HOM-C suggest many similarities in structure and functionwith the well-characterized Drosophila genes. Abdominal resemblesits Drosophila homolog abdominal-A in functioning to establishsegmental identities in the abdomen, such that in each casemutations result in homeotic transformations to PS6. Althoughthe anterior functional boundary of abdominal-A homologs isprecisely conserved, the domain within which Abdominal is importantextends more posterior than that of abdominal-A. The final expression pattern of the segmentation gene engrailedin Tribolium is identical to Drosophila, suggesting that thesehomologs are involved in a conserved developmental process.However, as expected the development of that pattern is different;engrailed stripes anticipate the formation of each new segmentas they appear sequentially in the elongating germ band. Althoughthe grasshopper even-skipped and fushi tarazu homologs are notapparently important in segmentation, the expression patternsof the Tribolium homologs strongly suggest that they have gaineda role in segmentation in the lineage leading to beetles andflies. Nevertheless, differences between Tribolium and Drosophilain the dynamics of even-skipped expression and the fushi tarazumutant phenotype indicate divergence in the regulation and rolesof these genes.     

Genetic Dissection of Cerebellar Development: Mutations Affecting Cell Position

SYNOPSIS. The spastic mutation induces swimming coordinationand equilibrium deficiencies in the Mexican axolotl (Ambystomamexicanum). Behavioral ontogeny studies determined that spaslksfail to develop behavior trains of sinusoidal flexures necessaryto mediate escape swimming at the time of onset of cerebellarfunction. Behavior analysis, after lesioning different cranialnerve roots and CNS areas in wild-type animals, confirmed the"behavioral focus" of the mutation to lie in the auricle orvestibulo-cerebellum. Single unit recordings in the cerebellarauricle and adjacent brainstem vestibular zone (area acoustico-lateralis)of mutants revealed a full complement of vestibular unit typesfound in wild-type. However, the gene appears to alter the physicallocation of vestibular units in both areas, including a ventral"translocation" of cerebellar units responding to sustainedipsilateral tilt. Correlated with this unit translocation, mutantPurkinje cells and allied afferent tracts are malpositionedventrally, i.e., "crowded" into an ectopic zone in the ventro-posteriorcerebellum. Studies on cerebellar structure at the time of onsetof spasticity (early feeding stage) confirmed the ventral malpositioningof cerebellar cells and fiber tracts seen in adults. In conjunctionwith these larval studies, mutant larvae injected with tritiatedthymidine during early cerebellogenesis and assayed at the earlyfeeding stage revealed a medio-ventral malpositioning of labelledcells; in wild-type, labelled cells were positioned laterally.Interestingly, the neuropathology of the reeler mutation (rev.,Mariani et al., 1977) found in the mouse is remarkably similarto that of the spastic axolotl. Both cerebella are reduced insize, misshapen, and lack fissures. Purkinje cells appear ectopicallyin the granular layer, white matter and deep cerebellar nuclei.Although both cerebella lack structural integrity, no individualcell type shows marked or progressive degeneration, and cellularuntrastructure appears intact; thus, both genes appear to actindependently of the genesis of cerebellar elements. Instead,they appear to influence morphogenetic movements by which presumptivecerebellar cells attain their proper positions during the neurogeneticsequence.     

Patterns of purine synthesis related to iridophore development in the wild type, melanoid, and axanthic strains of the Mexican axolotl, Ambystoma mexicanum Shaw

Differentiation of the iridophore in the axolotl is inhibited by each of two nonlinked autosomal recessive genes, melanoid (m) and axanthic (ax). Purines were extracted from wild type (M-Ax-), melanoid (mm), axanthic (axax), and melanoid-axanthic (mm axax) somatopleur. This tissue is rich in iridophores in wild-type axolotl larvae. Purines in ethanol and in water extracts from each genotype were separated by paper chromatography. Patterns with respect to the kinds of purines present were distinct for each genotype. Two compounds present in wild type were missing in both melanoid and axanthic mutants, which lack iridophores. One compound was present in wild type and melanoid, which have xanthophores, but was lacking in the axanthic mutant, which does not. The double homozygote (mm axax) lacked a purine found in wild type and each of the mutants singly. Possibly in this case two biosynthetic pathways may produce the same intermediate product. Thus the actions of the melanoid and axanthic genes in the axolotl are demonstrable chemically as differences in the kinds of purines present in the various genotypes.     

Myofibril-Inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts

The Mexican axolotl, Ambystoma mexicanum, carries the naturally-occurring recessive mutant gene 'c' that results in a failure of homozygous (c/c) embryos to form hearts that beat because of an absence of organized myofibrils. Our previous studies have shown that a noncoding RNA, Myofibril-Inducing RNA (MIR), is capable of promoting myofibrillogenesis and heart beating in the mutant (c/c) axolotls. The present study demonstrates that the MIR gene is essential for tropomyosin (TM) expression in axolotl hearts during development. Gene expression studies show that mRNA expression of various tropomyosin isoforms in untreated mutant hearts and in normal hearts knocked down with double-stranded MIR (dsMIR) are similar to untreated normal. However, at the protein level, selected tropomyosin isoforms are significantly reduced in mutant and dsMIR treated normal hearts. These results suggest that MIR is involved in controlling the translation or post-translation of various TM isoforms and subsequently of regulating cardiac contractility.