bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice
Authors: Yang Xiu-Ming , Vogan Kyle , Gros Philippe , Park Morag
Published: April 2021

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by wholemount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and medial tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both metand HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a subpopulation of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.
Redundant control of Ultrabithorax by zeste involves functional levels of zeste protein binding at the Ultrabithorax promoter
Authors: Laney Jeffrey D. , Biggin Mark D.
Published: April 2021

Many biological processes appear to be controlled by functionally redundant genes or pathways, but it has proven difficult to understand the nature of this redundancy. Here, we have analyzed a redundant regulatory interaction between the Drosophila transcription factor zeste and the homeotic gene Ultrabithorax. Mutations in zeste do not affect the cis-regulation of the endogenous Ultrabithorax gene; however, the expression of small Ultrabithorax promoter constructs is strongly dependent upon zeste. We show that this difference is due to redundant cis-regulatory elements in the Ultrabithorax gene, which presumably contain binding sites for factors that share the function of zeste. We also provide evidence suggesting that zeste and the gene encoding the GAGA factor have an overlapping function in regulating Ultrabithorax. Furthermore, we show that the zeste protein is bound at equal levels in vivo to a Ultrabithorax promoter construct, which zeste strongly activates, and to the identical promoter region in the endogenous Ultrabithorax gene, which zeste redundantly regulates. These results suggest that zeste is significantly active in the wild-type animal and not simply a factor that is induced as a back-up when other activators fail.
Variation in the number of activated torso receptors correlates with differential gene expression
Authors: Furriols Marc , Sprenger Frank , Casanova Jordi
Published: April 2021

Activation of receptor tyrosine kinases triggers many developmental decisions, yet we do not understand how activation of a single receptor can be transduced into different cell responses. The torso pathway in Drosophila provides a model to address this issue since it generates more than one response in the embryo. The torso receptor tyrosine kinase is activated at the embryonic poles under the control of trunk, a protein with similarities to several types of extracellular growth factors. Activation of torso is responsible for the development of a variety of structures, whose appearance can be correlated with activation of at least two different genes along the terminal region. In this study we have analyzed mutations in torso and trunk that express low levels of the respective proteins. We show that different amounts of torso or trunk molecules correlate with the expression of different zygotic genes, implicating changes in the number of activated torso molecules as one of the mechanisms defining differential gene expression. We suggest that variation in the number of activated receptors at the cell surface is a general mechanism that leads to differential gene expression and thus the generation of different cell responses.
Role of TGFβs and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod
Authors: Gañan Y. , Macias D. , Duterque-Coquillaud M. , Ros M. A. , Hurle J. M.
Published: April 2021

The establishment of the digital rays and the interdigital spaces in the developing limb autopod is accompanied by the occurrence of corresponding domains of expression of TGFβs and BMPs. This study analyzes whether these coincident events are functionally correlated. The experiments consisted of local administration of TGFβ-1, TGFβ-2 or BMP-4 by means of heparin or Affi-gel blue beads to the chick limb autopod in the stages preceding the onset of interdigital cell death. When beads bearing either TGFβ-1 or -2 were implanted in the interdigits, the mesodermal cells were diverted from the death program forming ectopic cartilages or extra digits in a doseand stagedependent fashion. This change in the interdigital phenotype was preceded by a precocious ectopic expression of ck-erg gene around the bead accompanied by down-regulation of bmp-4, msx-1 and msx-2 gene expression. When BMP-beads were implanted in the interdigital spaces, programmed cell death and the freeing of the digits were both accelerated. Implantation of beads bearing BMP-4 at the tip of the growing digits was followed by digit bifurcation, accompanied by the formation of an ectopic area of cell death resembling an extra interdigit, both morphologically and molecularly. The death-inducing effect of the BMP beads and the chondrogenic-inducing effect of the TGFβ beads were antagonized by the implantation of an additional bead preabsorbed with FGF-2, which constitutes a signal characteristic of the progress zone. It is concluded that the spatial distribution of digital rays and interdigital spaces might be controlled by a patterned distribution of TGFβs and BMPs in the mesoderm subjacent to the progress zone.
Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4
Authors: Ladher R. , Mohun T. J. , Smith J. C. , Snape A. M.
Published: April 2021

Bone morphogenetic protein-4 (BMP-4) is thought to play an important role in early Xenopus development by acting as a ‘ventralizing factor’ and as an epidermal determinant: local inhibition of BMP-4 function in whole embryos causes the formation of an additional dorsal axis, and inhibition of BMP-4 function in isolated ectodermal cells causes the formation of neural tissue. In this paper we describe a homeobox-containing gene whose expression pattern is similar to that of BMP-4, whose expression requires BMP-4 signalling and which, when over-expressed, causes a phenotype similar to that caused by over-expression of BMP-4. We suggest that this gene, which we call Xom, acts downstream of BMP-4 to mediate its effects.
The SM 22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice
Authors: Moessler H. , Mericskay M. , Li Z. , Nagl S. , Paulin D. , Small J. V.
Published: April 2021

The transcriptional signals underlying smooth muscle differentiation are currently unknown. We report here the complete sequence and characterization of the single mouse gene for the smooth muscle-specific protein SM 22 and the transcriptional activity of its promoter in cultured smooth muscle cells in vitro and in transgenic mice. In the transgenic animals, promoter constructs ranging in length from 445 to 2126 bp directed reporter expression initially in the heart and the somites of embryos and subsequently in the arteries of the vascular system, but in none of the visceral muscles, nor in the veins. Expression in the heart was spatially restricted to the presumptive right ventricle and outflow tract and disappeared in the adult. Likewise, expression in the somites was only transitory and was not observed after about 14.5 days post coitum in the embryo. In the adult mouse, SM 22 promoter activity persisted in the smooth muscle cells of the arteries and was still notably absent from other smooth muscles, despite the ubiquitous presence of the endogenous SM 22 protein. These findings on the transcriptional activity of a smooth muscle promoter in vivo reveal the existence of different differentiation programmes for smooth muscle cells in the veins and the arteries and raise the expectation of a further subdivision of programmes among the visceral muscles.
Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm
Authors: Conlon F. L. , Sedgwick S. G. , Weston K. M. , Smith J. C.
Published: April 2021

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.
The intracellular deletions of DELTA and SERRATE define dominant negative forms of the Drosophila Notch ligands
Authors: Sun Xin , Artavanis-Tsakonas Spyros
Published: April 2021

We examined the function of the intracellular domains of the two known Drosophila Notch ligands, Delta and Serrate, by expressing wild-type and mutant forms in the developing Drosophila eye under the sevenless promoter. The expression of intracellularly truncated forms of either Delta (sev-DlTM) or Serrate (sev-SerTM) leads to extra photoreceptor phenotypes, similar to the eye phenotypes associated with loss-of-function mutations of either Notch or Delta. Consistent with the notion that the truncated ligands reduce Notch signalling activity, the eye phenotypes of sevDlTM and sev-SerTM are enhanced by loss-of-function mutations in the Notch pathway elements, Notch, Delta, mastermind, deltex and groucho, but are suppressed by a duplication of Delta or mutations in Hairless, a negative regulator of the pathway. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mδ, a target of Notch signalling, is down-regulated by the truncated ligands highly expressed in neighbouring cells. We conclude that the truncated ligands act as antagonists of Notch signalling.
Inhibition of floor plate differentiation by Pax3: evidence from ectopic expression in transgenic mice
Authors: Tremblay Patrick , Pituello Fabienne , Gruss Peter
Published: April 2021

The Pax genes containing a complete paired-type homeobox are expressed in restricted dorsoventral domains of the undifferentiated neuroepithelium. Their expression respond to signals that pattern the neural tube and which emanate from the notochord, floor plate and overlying ectoderm. In order to determine whether the dorsally restricted Pax3 gene can influence the fate of cells within the neural tube, we produced transgenic mice overexpressing Pax3 in the entire neural tube under the Hoxb4 promoter/region A enhancer. In two distinct transgenic mouse lines, we observed embryos with abnormal limb, eye, brain and neural tube development. Expression of Pax3 was not sufficient to dorsalize cells from the ventral intermediate zone, which still expressed ventral markers. However, in these embryos, expression of Pax3 at the ventral midline was associated with the absence of floor plate differentiation. Under these conditions, motor neurons still differentiated in the ventral spinal cord, although in fewer numbers.
Mutations affecting development of the zebrafish retina
Authors: Malicki Jarema , Neuhauss Stephan C. F. , Schier Alexander F. , Solnica-Krezel Lilianna , Stemple Derek L. , Stainier Didier Y. R , Abdelilah Salim , Zwartkruis Fried , Rangini Zehava , Driever Wolfgang
Published: April 2021

In a large scale screen for genetic defects in zebrafish embryogenesis we identified 49 mutations affecting development of the retina. Based on analysis of living embryos as well as histological sections, we grouped the isolated mutations into six phenotypic categories. (1) Mutations in three loci result in a loss of wild-type laminar pattern of the neural retina. (2) Defects in four loci lead to an abnormal specification of the eye anlagen. Only one eye frequently forms in this class of mutants. (3) Seven loci predominantly affect development of the outer retinal layers. Mutants in this category display cell loss mainly in the photoreceptor cell layer. (4) Nine mutations cause retardation of eye growth without any other obvious abnormalities in the retina. (5) A group of twelve mutations is characterized by nonspecific retinal degeneration. (6) Four mutations display retinal degeneration associated with a pigmentation defect. Finally, two mutations, one with absence of the ventral retina and one with an eye-specific pigmentation defect, are not classified in any of the above groups. The identified mutations affect numerous aspects of eye development, including: specification of the eye anlage, growth rate of the optic cup, establishment of retinal stratification, specification or differentiation of retinal neurons and formation of the dorsoventral axis in the developing eye.
Mutations affecting development of the zebrafish ear
Authors: Malicki Jarema , Schier Alexander F. , Solnica-Krezel Lilianna , Stemple Derek L. , Neuhauss Stephan C. F. , Stainier Didier Y. R. , Abdelilah Salim , Rangini Zehava , Zwartkruis Fried , Driever Wolfgang
Published: April 2021

In a large scale screen for genetic defects in zebrafish embryogenesis we identified mutations affecting several aspects of ear development, including: specification of the otic placode, growth of the otic vesicle (otocyst), otolith formation, morphogenesis of the semicircular canals and differentiation of the otic capsule. Here we report initial phenotypic and genetic characterization of 20 of these mutations defining 13 independent loci. Embryos mutant at the quadro locus display abnormal specification of the otic placode. As revealed by dlx-3 expression, the otic field in the mutant embryos is smaller or split into two fields. At later stages of development the ear of quadro mutants is frequently divided into two smaller, incomplete units. Four loci affect ear shape shortly after formation of the otic vesicle. All of them also display abnormal brain morphology. Mutations in five loci result in the absence of otolith formation; two of these also produce changes of ear morphology. Two loci, little richard and golas, affect morphology of the otic vesicle shortly before formation of the semicircular canals. In both cases the morphogenesis of the semicircular canals is disrupted. Finally, the antytalent locus is involved in late expansion of the ear structure. Analysis of mutations presented here will strengthen our understanding of vertebrate ear morphogenesis and provide novel entry points to its genetic analysis.
Hematopoietic mutations in the zebrafish
Authors: Weinstein Brant M. , Schier Alexander F. , Abdelilah Salim , Malicki Jarema , Solnica-Krezel Lilliana , Stemple Derek L. , Stainier Didier Y. R. , Zwartkruis Fried , Driever Wolfgang , Fishman Mark C.
Published: April 2021

We have identified mutations that perturb the formation or differentiation of the first embryonic blood cells in the zebrafish embryo. These ‘primitive’ red blood cells originate in the intermediate cell mass of the trunk, a derivative of the dorsal lateral plate mesoderm. By transfusion of blood between embryos we demonstrate that this cohort of cells provides the embryo with all, or nearly all, of its blood cells until at least day 5 postfertilization. Larval lethal mutations generated by ENU mutagenesis affect different steps in the development of these cells. Some cause defects in precursor generation, others defects in differentiation, and others an increase in cellular photosensitivity.
Characterization of zebrafish mutants with defects in embryonic hematopoiesis
Authors: Ransom David G. , Haffter Pascal , Odenthal Jörg , Brownlie Alison , Vogelsang Elisabeth , Kelsh Robert N. , Brand Michael , Eeden Fredericus J. M. van , Furutani-Seiki Makoto , Granato Michael , Hammerschmidt Matthias , Heisenberg Carl-Phillip , Jiang Yun-Jin , Kane Donald A. , Mullins Mary C. , Nüsslein-Volhard Christiane
Published: April 2021

As part of a large scale chemical mutagenesis screen of the zebrafish (Danio rerio) genome, we have identified 33 mutants with defects in hematopoiesis. Complementation analysis placed 32 of these mutants into 17 complementation groups. The allelism of the remaining 1 blood mutant is currently unresolved. We have categorized these blood mutants into four phenotypic classes based on analyses of whole embryos and isolated blood cells, as well as by in situ hybridization using the hematopoietic transcription factors GATA-1 and GATA-2. Embryos mutant for the gene moonshine have few if any proerythroblasts visible on the day circulation begins and normal erythroid cell differentiation is blocked as determined by staining for hemoglo-bin and GATA-1 expression. Mutations in five genes, chablis, frascati, merlot, retsina, thunderbird and two possibly unique mutations cause a progressive decrease in the number of blood cells during the first 5 days of development. Mutations in another seven genes, chardonnay, chianti, grenache, sauternes, weißherbst and zinfandel, and two additional mutations result in hypochromic blood cells which also decrease in number as development proceeds. Several of these mutants have immature cells in the circulation, indicating a block in normal erythroid development. The mutation in zinfandel is dominant, and 2-day old heterozygous carriers fail to express detectable levels of hemoglobin and have decreasing numbers of circulating cells during the first 5 days of development. Mutations in two genes, freixenet and yquem, result in the animals that are photosensitive with autofluorescent blood, similar to that found in the human congenital porphyrias. The collection of mutants presented here represent several steps required for normal erythropoiesis. The analysis of these mutants provides a powerful approach towards defining the molecular mechanisms involved in vertebrate hematopoietic development.
Jaw and branchial arch mutants in zebrafish I: branchial arches
Authors: Schilling Thomas F. , Piotrowski Tatjana , Grandel Heiner , Brand Michael , Heisenberg Carl-Philipp , Jiang Yun-Jin , Beuchle Dirk , Hammerschmidt Matthias , Kane Donald A. , Mullins Mary C. , Eeden Fredericus J. M. van , Kelsh Robert N. , Furutani-Seiki Makoto , Granato Michael , Haffter Pascal , Odenthal Jörg , Warga Rachel M. , Trowe Torsten , Nüsslein-Volhard Christiane
Published: April 2021

Jaws and branchial arches together are a basic, segmented feature of the vertebrate head. Seven arches develop in the zebrafish embryo (Danio rerio), derived largely from neural crest cells that form the cartilaginous skeleton. In this and the following paper we describe the phenotypes of 109 arch mutants, focusing here on three classes that affect the posterior pharyngeal arches, including the hyoid and five gill-bearing arches. In lockjaw, the hyoid arch is strongly reduced and subsets of branchial arches do not develop. Mutants of a large second class, designated the flathead group, lack several adjacent branchial arches and their associated cartilages. Five alleles at the flathead locus all lead to larvae that lack arches 4-6. Among 34 other flathead group members complementation tests are incomplete, but at least six unique phenotypes can be distinguished. These all delete continuous stretches of adjacent branchial arches and unpaired cartilages in the ventral midline. Many show cell death in the midbrain, from which some neural crest precursors of the arches originate. lockjaw and a few mutants in the flathead group, including pistachio, affect both jaw cartilage and pigmentation, reflecting essential functions of these genes in at least two neural crest lineages. Mutants of a third class, including boxer, dackel and pincher, affect pectoral fins and axonal trajectories in the brain, as well as the arches. Their skeletal phenotypes suggest that they disrupt cartilage morphogenesis in all arches. Our results suggest that there are sets of genes that: (1) specify neural crest cells in groups of adjacent head segments, and (2) function in common genetic pathways in a variety of tissues including the brain, pectoral fins and pigment cells as well as pharyngeal arches.
Mutations affecting craniofacial development in zebrafish
Authors: Neuhauss Stephan C. F. , Solnica-Krezel Lilianna , Schier Alexander F. , Zwartkruis Fried , Stemple Derek L. , Malicki Jarema , Abdelilah Salim , Stainier Didier Y. R. , Driever Wolfgang
Published: April 2021

In a large-scale screen for mutations affecting embryogenesis in zebrafish, we identified 48 mutations in 34 genetic loci specifically affecting craniofacial development. Mutants were analyzed for abnormalities in the cartilaginous head skeleton. Further, the expression of marker genes was studied to investigate potential abnormalities in mutant rhombencephalon, neural crest, and pharyngeal endoderm. The results suggest that the identified mutations affect three distinct aspects of craniofacial development. In one group, mutations affect the overall pattern of the craniofacial skeleton, suggesting that the genes are involved in the specification of these elements. Another large group of mutations affects differentiation and morphogenesis of cartilage, and may provide insight into the genetic control of chondrogenesis. The last group of mutations leads to the abnormal arrangement of skeletal elements and may uncover important tissue-tissue interactions underlying jaw development.
Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva
Authors: Granato Michael , Eeden Fredericus J. M. van , Schach Ursula , Trowe Torsten , Brand Michael , Furutani-Seiki Makoto , Haffter Pascal , Hammerschmidt Matthias , Heisenberg Carl-Philipp , Jiang Yun-Jin , Kane Donald A. , Kelsh Robert N. , Mullins Mary C. , Odenthal Jörg , Nüsslein-Volhard Christiane
Published: April 2021

Zebrafish embryos and larvae have stage-specific patterns of motility or locomotion. Two embryonic structures accomplish this behavior: the central nervous system (CNS) and skeletal muscles. To identify genes that are functionally involved in mediating and controlling different patterns of embryonic and larval motility, we included a simple touch response test in our zebrafish large-scale genetic screen. In total we identified 166 mutants with specific defects in embryonic motility. These mutants fall into 14 phenotypically distinct groups comprising at least 48 genes. Here we describe the various phenotypic groups including mutants with no or reduced motility, mechanosensory defective mutants, ‘spastic’ mutants, circling mutants and motor circuit defective mutants.

In 63 mutants, defining 18 genes, striation of somitic muscles is reduced. Phenotypic analysis provides evidence that these 18 genes have distinct and consecutive functions during somitic muscle development. The genes sloth (slo) and frozen (fro) already act during myoblast differentiation, while 13 genes appear to function later, in the formation of myofibers and the organization of sarcomeres. Mutations in four other genes result in muscle-specific degeneration.

103 mutations, defining at least 30 genes, cause no obvious defects in muscle formation and may instead affect neuronal development. Analysis of the behavioral defects suggests that these genes participate in the diverse loco-motion patterns observed, such as touch response, rhythmic tail movements, equilibrium control, or that they simply confer general motility to the animal. In some of these mutants specific defects in the developing nervous system are detected. Mutations in two genes, nevermind (nev) and macho (mao), affect axonal projection in the optic tectum, whereas axon formation and elongation of motorneurons are disrupted by mutations in the diwanka (diw) and the unplugged (unp) genes.
Specification of the embryonic limb primordium by graded activity of Decapentaplegic
Authors: Goto Satoshi , Hayashi Shigeo
Published: April 2021

Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. We show that Wingless, but not Decapentaplegic, is responsible for initial specification of the limb primordia with a distal identity. Limb formation is restricted to the lateral position of the embryo by negative control of the early function of Decapentaplegic and the EGF receptor homolog that determine the global dorsoventral pattern. Late function of Decapentaplegic locally determines two additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, which is in contrast to the consequence of decapentaplegic mutations in the imaginal disc, which cause a deletion of distal structures. The results indicate that the limb pattern elements are added in a distal to proximal direction in the embryo, which is opposite to what is happening in the growing imaginal disc. We propose that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis.
Planarian Hox genes: novel patterns of expression during regeneration
Authors: Bayascas J. R. , Castillo E. , Muñoz-Mármol A. M. , Saló E.
Published: April 2021

Platyhelminthes are widely considered to be the sister group of coelomates (Philippe, H., Chenuil, A. and Adoutte, A. (1994)Development 1994 Supplement, 15-24) and the first organisms to show bilateral symmetry and cephalization. Within this phylum, the freshwater planarians (Turbellaria, Tricladida) have been used as model systems for studying bidirectional regeneration (Slack, J. M. W. (1980) J. Theor. Biol. 82, 105-140). We have been attempting to identify potential pattern-control genes involved in the regeneration of planarian heads and tails after amputation. Since Hox cluster genes determine positional identity along the anteroposterior axis in a wide range of animals (Slack, J. M. W., Holland, P. W. H. and Graham, C. F. (1993) Nature 361, 490-492), we performed an extensive search for Hox-related genes in the planarian Dugesia(G)tigrina. Sequence analyses of seven planarian Dthox genes (Dthox-A to Dthox-G) reveal high similarities with the homeodomain region of the Hox cluster genes, allowing us to assign planarian Dthox genes to anterior and medial Hox cluster paralogous groups. Whole-mount in situ hybridization studies in regenerating adults showed very early, synchronous and colocalized activation of Dthox-D, Dthox-A, Dthox-C, Dthox-E, Dthox-G and Dthox-F. After one hour of regeneration a clear expression was observed in all Dthox genes studied. In addition, all seemed to be expressed in the same regenerative tissue, although in the last stages of regeneration (9 to 15 days) a differential timing of deactivation was observed. The same Dthox genes were also expressed synchronously and were colocalized during intercalary regeneration, although their expression was delayed. Terminal regeneration showed identical Dthox gene expression in anterior and posterior blastemas, which may prevent these genes from directing the distinction between head and tail. Finally, continuous expression along the whole lateral blastema in sagittal regenerates reflected a ubiquitous Dthox response in all types of regeneration that was not related specifically with the anteroposterior polarity.
Sclerotome development and peripheral nervous system segmentation in embryonic zebrafish
Authors: Morin-Kensicki Elizabeth M. , Eisen Judith S.
Published: April 2021

Vertebrate embryos display segmental patterns in many trunk structures, including somites and peripheral nervous system elements. Previous work in avian embryos suggests a role for somite-derived sclerotome in segmental patterning of the peripheral nervous system. We investigated sclerotome development and tested its role in patterning motor axons and dorsal root ganglia in embryonic zebrafish. Individual somite cells labeled with vital fluorescent dye revealed that some cells of a ventromedial cell cluster within each somite produced mesenchymal cells that migrated to positions expected for sclerotome. Individual somites showed anterior/posterior distinctions in several aspects of development: (1) anterior ventromedial cluster cells produced only sclerotome, (2) individual posterior ventromedial cluster cells produced both sclerotome and muscle, and (3) anterior sclerotome migrated earlier and along a more restricted path than posterior sclerotome. Vital labeling showed that anterior sclerotome colocalized with extending identified motor axons and migrating neural crest cells. To investigate sclerotome involvement in peripheral nervous system patterning, we ablated the ventromedial cell cluster and observed subsequent development of peripheral nervous system elements. Primary motor axons were essentially unaffected by sclerotome ablation, although in some cases outgrowth was delayed. Removal of sclerotome did not disrupt segmental pattern or development of dorsal root ganglia or peripheral nerves to axial muscle. We propose that peripheral nervous system segmentation is established through interactions with adjacent paraxial mesoderm which develops as sclerotome in some vertebrate species and myotome in others.
The Drosophila dCREB-A gene is required for dorsal/ventral patterning of the larval cuticle
Authors: Andrew Deborah J. , Baig Anjum , Bhanot Purnima , Smolik Sarah M. , Henderson Katya D.
Published: April 2021

We report on the characterization of the first loss-of-function mutation in a Drosophila CREB gene, dCREB-A. In the epidermis, dCREB-A is required for patterning cuticular structures on both dorsal and ventral surfaces since dCREB-A mutant larvae have only lateral structures around the entire circumference of each segment. Based on results from epistasis tests with known dorsal/ventral patterning genes, we propose that dCREB-A encodes a transcription factor that functions near the end of both the DPP- and SPI-signaling cascades to translate the corresponding extracellular signals into changes in gene expression. The lateralizing phenotype of dCREB-A mutants reveals a much broader function for CREB proteins than previously thought.
Compartmental organization of the Drosophila genital imaginal discs
Authors: Chen Elizabeth H. , Baker Bruce S.
Published: April 2021

We have investigated the anterior and posterior compartmental organization of the genital imaginal disc. Unlike the thoracic discs, the genital disc is a compound disc consisting of three primordia – the female genital, male genital, and anal primordia. Here we provide evidence that each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in compartment-specific manners in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments. Furthermore, we examined the phenotypes of mutant clones of the cAMP-dependent protein kinase A and engrailed-invected, genes that are known to play compartment-specific functions in other discs. These experiments demonstrate that the anterior/posterior patterning functions of these genes are conserved in the genital disc. The adult clonal phenotypes of protein kinase A and engrailed-invected mutants also provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. Our results lead us to propose a new model to describe the anterior and posterior compartmental organization of the genital disc.
Sperm-egg fusion is the prelude to the initial Ca2+ increase at fertilization in the mouse
Authors: Lawrence Yvonne , Whitaker Michael , Swann Karl
Published: April 2021

Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+]i oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initated by events that are closely coupled to the fusion of the sperm and egg membranes.
Evolutionary changes of developmental mechanisms in the absence of cell lineage alterations during vulva formation in the Diplogastridae (Nematoda)
Authors: Sommer Ralf J.
Published: April 2021

The origin of novelty is one of the least understood evolutionary phenomena. One approach to study evolutionary novelty comes from developmental biology. During developmental cell fate specification of the nematode Pristionchus pacificus (Diplogastridae), five cell fates can be distinguished within a group of twelve ventral epidermal cells. The differentiation pattern of individual cells includes programmed cell death, cell fusion and vulval differentiation after induction by the gonad. A cell lineage comparison among species of seven different genera of the Diplogastridae indicates that the differentiation pattern of ventral epidermal cells is highly conserved. Despite this morpho-logical conservation, cell ablation experiments indicate many independent alterations of underlying mechanisms of cell fate specification. Cell fusion and individual cell competence change during evolution as well as the differentiation property in response to inductive signaling. These results suggest that developmental mechanisms, some of which are redundantly involved in vulval fate specification of the genetic model organism Caenorhabditis elegans, can evolve without concomitant morphological change.
Two nested gonadal inductions of the vulva in nematodes
Authors: Félix Marie-Anne , Sternberg Paul W.
Published: April 2021

How do intercellular signals that pattern cell fates vary in evolution? During nematode vulva development, precursor cells acquire one of three fates in a pattern centered around the gonadal anchor cell. Non-vulval fates are at the periphery, outer and inner vulval fates are towards the center. In Caenorhabditis elegans, the three fates are specified around the same time by an induction by the anchor cell and lateral signaling between the vulva precursor cells. We find that, in three other nematode species (Panagrolaimus, Oscheius and Rhabditella spp.) spanning two families, the centered pattern is obtained by two temporally distinct gonadal inductions. The first induction specifies vulval fates; the second induction specifies the inner vulval fates in a subset of the precursors’ daughters. This evolutionary change in the spatiotemporal connectivity of cell interactions allows centering of the pattern between two precursors in Panagrolaimus.
Organization and cell differentiation in lateral roots of Arabidopsis thaliana
Authors: Malamy Jocelyn E. , Benfey Philip N.
Published: April 2021

Lateral root formation in plants involves the stimulation of mature pericycle cells to proliferate and redifferentiate to create a new organ. The simple organization of the root of Arabidopsis thaliana allows the development of lateral root primordia to be characterized histologically. We have divided the process of lateral root development into 8 stages defined by specific anatomical characteristics and cell divisions. To identify the cell types in the developing primordium we have generated a collection of marker lines that express β-glucuronidase in a tissue- or cell type-specific manner in the root. Using these tools we have constructed a model describing the lineage of each cell type in the lateral root. These studies show that organization and cell differentiation in the lateral root primordia precede the appearance of a lateral root meristem, with differential gene expression apparent after the first set of divisions of the pericycle.
Long-range genomic rearrangements upstream of Kit dysregulate the developmental pattern of Kit expression in W
57 and W
banded mice and interfere with distinct steps in melanocyte development
Authors: Klüppel Michael , Nagle Deborah L. , Bucan Maja , Bernstein Alan
Published: April 2021

Mutations in the murine dominant white spotting (W) locus cause pleiotropic developmental defects that affect hematopoietic cells, melanocytes, germ cells and the interstitial cells of Cajal in the gut. W mutations either alter the coding sequence of the Kit receptor tyrosine kinase, resulting in a receptor with impaired kinase activity, or affect Kit expression. Here we describe the molecular and cell-type-specific developmental defects of two of the latter class of regulatory W alleles, W57 and Wbanded(bd). In both mutants, the temporal and spatial patterns of Kit expression are dysregulated during embryogenesis and in adult animals. In Wbd mice, ectopic expression of Kit in the dermatome of the somites at days 10.8 and 11.8 of development seemed to interfere with melanoblast development. In contrast, the W57 allele leads to an intrinsic pigmentation defect by downregulating developmental Kit expression in trunk melanoblasts, but not melanoblasts around the otic vesicle. Both mutations affect transcriptional initiation of the Kit gene. The W57 allele is associated with a 80 kb deletion 5′ of the Kit-coding region while Wbd is associated with a 2.8 Mb genomic inversion of chromosome 5 with the distal breakpoint between Kit and the platelet-derived growth factor receptor alpha (Pdgfra) gene, and the proximal breakpoint between the genes for the GABA receptor beta 1 (Gabrb1) and the Tec tyrosine kinase, juxtaposing the Kit and Tec tyrosine kinase genes. Neither W57 nor Wbd affect genomic sequences previously suggested in in vitro experiments to control cell-type-specific expression of Kit. These results link specific mechanisms of cellular and developmental defects to long-range genomic rearrangements that positively and negatively affect Kit transcription in different cell lineages as well as in different subpopulations of the same lineage.
Fashioning the vertebrate heart: earliest embryonic decisions
Authors: Fishman Mark C. , Chien Kenneth R.
Published: April 2021

Our goal here is to set out the types of unitary decisions made by heart progenitor cells, from their appearance in the heart field until they form the simple heart tube. This provides a context to evaluate cell fate, lineage and, finally, morphogenetic decisions that configure global heart form and function. Some paradigms for cellular differentiation and for pattern generation may be borrowed from invertebrates, but neither Drosophila nor Caenorhabditis elegans suffice to unravel higher order decisions. Genetic analyses in mouse and zebrafish may provide one entrance to these pathways.
Analysis of competence and of Brachyury autoinduction by use of hormoneinducible Xbra
Authors: Tada M. , O’Reilly M.-A. J. , Smith J. C.
Published: April 2021

Analysis of gene function in Xenopus development frequently involves over-expression experiments, in which RNA encoding the protein of interest is microinjected into the early embryo. By taking advantage of the fate map of Xenopus, it is possible to direct expression of the protein to particular regions of the embryo, but it has not been possible to exert control over the timing of expression; the protein is translated immediately after injection. To overcome this problem in our analysis of the role of Brachyury in Xenopus development, we have, like Kolm and Sive (1995; Dev. Biol. 171, 267-272), explored the use of hormone-inducible constructs. Animal pole regions derived from embryos expressing a fusion protein (Xbra-GR) in which the Xbra open reading frame is fused to the ligandbinding domain of the human glucocorticoid receptor develop as atypical epidermis, presumably because Xbra is sequestered by the heat-shock apparatus of the cell. Addition of dexamethasone, which binds to the glucocorticoid receptor and releases Xbra, causes formation of mesoderm. We have used this approach to investigate the competence of animal pole explants to respond to Xbra-GR, and have found that competence persists until late gastrula stages, even though by this time animal caps have lost the ability to respond to mesoderm-inducing factors such as activin and FGF. In a second series of experiments, we demonstrate that Xbra is capable of inducing its own expression, but that this auto-induction requires intercellular signals and FGF signalling. Finally, we suggest that the use of inducible constructs may assist in the search for target genes of Brachyury.
Zfx mutation results in small animal size and reduced germ cell number in male and female mice
Authors: Luoh Shiuh-Wen , Bain Paul A. , Polakiewicz Roberto D. , Goodheart Mary L. , Gardner Humphrey , Jaenisch Rudolf , Page David C.
Published: April 2021

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been spec-ulated to function in sex differentiation, spermatogenesis, and Turner syndrome. We derived Zfx mutant mice by targeted mutagenesis. Mutant mice (both males and females) were smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals were fully masculinized, with testes and male genitalia, and were fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals were fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells was reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibited a growth deficit evident at embryonic day 12.5, which persisted throughout postnatal life and was not complemented by the Zfy genes. These phe-notypes provide the first direct evidence for a role ofZfx in growth and reproductive development.
Hedgehog is an indirect regulator of morphogenetic furrow progression in the Drosophila eye disc
Authors: Strutt David I. , Mlodzik Marek
Published: April 2021

Pattern formation in the eye imaginal disc of Drosophila occurs in a wave that moves from posterior to anterior. The anterior edge of this wave is marked by a contracted band of cells known as the morphogenetic furrow, behind which photoreceptors differentiate. The movement of the furrow is dependent upon the secretion of the signalling protein Hedgehog (Hh) by more posterior cells, and it has been suggested that Hh acts as an inductive signal to induce cells to enter a furrow fate and begin differentiation. To further define the rôle of Hh in this process, we have analysed clones of cells lacking the function of the smoothened (smo) gene, which is required for transduction of the Hh signal and allows the investigation of the autonomous requirement for hh signalling. These experiments demonstrate that the function of hh in furrow progression is indirect. Cells that cannot receive/transduce the Hh signal are still capable of entering a furrow fate and differentiating normally. However, hh is required to promote furrow progression and regulate its rate of movement across the disc, since the furrow is significantly delayed in smo clones.