HH Stages Research Articles

High-Performance Skutterudite/Half-Heusler Cascaded Thermoelectric Module Using the Transient Liquid Phase Sintering Joining Technique.

Thermoelectric (TE) materials have made rapid advancement in the past decade, paving the pathway toward the design of solid-state waste heat recovery systems. The next requirement in the design process is realization of full-scale multistage TE devices in the medium to high temperature range for enhanced power generation. Here, we report the design and manufacturing of full-scale skutterudite (SKD)/half-Heusler (hH) cascaded TE devices with 49-couple TE legs for each stage. The automated pick-and-place tool is employed for module fabrication providing overall high manufacturing process efficiency and repeatability. Optimized Ti/Ni/Au coating layers are developed for metallization as the diffusion barrier and electrode contact layers. The Cu-Sn transient liquid phase sintering technique is utilized for SKD and hH stages, which provides a high strength bonding and very low contact resistance. A remarkably high output power of 38.3 W with a device power density of 2.8 W·cm-2 at a temperature gradient of 513 °C is achieved. These results provide an avenue for widespread utilization of TE technology in waste heat recovery applications.

Development of the chicken skull: A complement to the external staging table of Hamburger and Hamilton.

Embryonic staging tables provide a standard of developmental stages that can be used by individual investigators and provide approximate time points for the study of developmental phenomena. Surprisingly, despite the presence of a plethora of studies on the chicken skull and its role as a model species in developmental research, a staging table of the development of the chicken skull remains lacking. A detailed photographic staging table of the osseous portion of the chicken skull is thus presented here based on cleared and stained HH stages spanning HH 35 (first appearance of skull ossification) to the final stage before hatching (HH 45). This table documents the development of most of the cranial elements in the skull from the start of ossification until the element takes its final shape. The table shows that the elements of the lower jaw and ventral side of the skull begin ossifying before the skull roof and that most elements take roughly 5 days to reach their final shape, whereas others take up to 9 days (e.g., the frontal). The obtained results lead to several hypotheses about chicken skull development and provide a timeframe for future studies on chicken skull development.

In vitro improvement of quail primordial germ cell expansion through activation of TGF-beta signaling pathway.

Avian primordial germ cells (PGCs) have valuable potentials to cell-based approaches for transgenic bird production. In this regard, improvement of avian PGC expansion in vitro is necessary. Among experimental avian species, quail is a good model for transgenic technology, especially due to its short generation time. In the present study, we have examined the proliferative effects of transforming growth factor β (TGF-β) on the quail PGCs. After isolation of quail PGCs from blood (Hamburger-Hamilton [HH stages 13-15]) and gonads (HH stages 28-30), these cells were cultured on quail embryonic fibroblasts (QEF). Our results indicated th at cultured gonadal-derived PGCs proliferated 400 times in comparison to 100 times for blood PGCs over 40-50 days. Upon in vitro exposure to TGF-β inducers by Activin or the inducer of definitive endoderm 1 (IDE1) small molecule, the number of gonad PGCs significantly increased to 26% and 64%, respectively. In contrast, inhibition of the TGF-β signaling pathway by SB431542 resulted in a significant reduction in the numbers of PGCs (P < 0.001). Moreover, Phosphorylation of SMAD2/3 in the IDE1 group was higher compared to the Activin-treated ones. We confirmed the PGC identification with periodic acid-Schiff (PAS) staining, anti-SSEA1, β-catenin, β-integrin, and Nanog immunofluorescence staining. Exogenously IDE1 treated-PGCs migrated toward the embryonic gonads after transplantation into the heart of the recipient embryo at HH stages 13-15. Our results suggested that the application of IDE1 small molecule into the culture of quail PGCs represented a step toward achieving efficient expansion of the avian PGCs.

Neurosphere formation potential resides not in the caudal cell mass, but in the secondary neural tube.

The caudal cell mass (CCM) is known as the main player in secondary neurulation, forming the secondary neural tube (2NT). This suggests that the CCM may have the character of neural progenitor cells. The neural potential of the CCM and the 2NT (CCM + 2NT) was assessed by in vitro culture of neurospheres during Hamburger and Hamilton stages (HH) of secondary neurulation (HH16 to HH32). We also analyzed the neural potential of the developing central nervous system (CNS) by comparing the neurosphere culture from the brain, upper / lower spinal cord, and CCM + 2NT from various HH stages. The CCM + 2NT was capable of forming neurospheres. Of the various HH stages, the greatest number of neurospheres from CCM + 2NT were cultured at HH28. Because the 2NT is most prominent at HH28, we hypothesized that the 2NT, rather than the CCM, had the main potential to produce neurospheres. When the neurospheres were cultured separately from the CCM and the 2NT, 2NT made significantly more neurospheres. When comparing different parts of the CNS, at HH16, the greatest number of neurospheres was formed from the brain. At HH32, it was the CCM + 2NT. The region with the greatest number of neurospheres progressed in a cephalo-caudal direction during development. This study showed that neurospheres can be cultured from CCM + 2NT, and the main player in neurosphere formation was the 2NT. The present study has also revealed cephalo-caudal trend in the neural potential of developing CNS.

Transcription of subtelomere tandemly repetitive DNA in chicken embryogenesis.

Transcription of tandemly repetitive DNA in embryogenesis seems to be of special interest due to a crucial role of non-coding RNAs in many aspects of development. However, only a few data are available on tandem repeats transcription at subtelomere regions of chromosomes during vertebrate embryogenesis. To reduce this gap, we examined stage and tissue-specific pattern of subtelomeric PO41 (pattern of 41 bp) tandem repeat transcription during embryogenesis of chicken (Gallus gallus domesticus). Using whole-mount RNA fluorescent in situ hybridization and reverse transcription PCR with specific primers, we demonstrated that both strands of PO41 repeat are transcribed at each of the studied stages of chicken embryo development: from 7-8 HH to 20 HH stages. Subtelomere-derived transcripts localize in the nuclei of all cell types and throughout the all embryonic bodies: head, somites, tail, wings and buds. In embryo-dividing cells and cultured embryonic fibroblasts, PO41 RNAs envelop terminal regions of chromosomes. PO41-containing RNAs are predominantly single-stranded and can be polyadenylated, indicating appearance of non-nascent form of subtelomeric transcripts. PO41 repeat RNAs represent a rare example of ubiquitously transcribed non-coding RNAs, such as Xist/XIST RNA or telomere repeat-containing RNA. Distribution of PO41 repeat transcripts at different stages of embryo development and among cell types has extremely uniform pattern, indicating on possible universal functions of PO41 non-coding RNAs.

Spatio-Temporally Restricted Expression of Cell Adhesion Molecules during Chicken Embryonic Development

Differential cell adhesive properties are known to regulate important developmental events like cell sorting and cell migration. Cadherins and protocadherins are known to mediate these cellular properties. Though a large number of such molecules have been predicted, their characterization in terms of interactive properties and cellular roles is far from being comprehensive. To narrow down the tissue context and collect correlative evidence for tissue specific roles of these molecules, we have carried out whole-mount in situ hybridization based RNA expression study for seven cadherins and four protocadherins. In developing chicken embryos (HH stages 18, 22, 26 and 28) cadherins and protocadherins are expressed in tissue restricted manner. This expression study elucidates precise expression domains of cell adhesion molecules in the context of developing embryos. These expression domains provide spatio-temporal context in which the function of these genes can be further explored.

Physical lineage tracing of cells contributing to the chicken cardiac conduction system

Purpose: Knowledge about the developmental origin of the cardiac conduction system (CCS) is important to understand the pathophysiology of arrhythmias. During early development, the heart consists of two bilateral plates of splanchnic mesoderm, which fuse to form the primary heart tube (PHT) derived from the first heart field. A large number of cells at the venous pole of the PHT constitute a different cardiac progenitor population, called the second heart field (SHF). This population of cells expresses the LIM-homeodomain transcription factor Islet-1 (Isl-1). The contribution of this SHF population to the developing CCS remains the subject of discussion. We explored the feasibility of in ovo physical lineage tracing in chicken embryos, to perform long-term follow-up of SHF-derived cells contributing to the CCS. Methods and results: Labeling was achieved by injecting a solution of two fluorescent dyes (DiI/5-TAMRA, Invitrogen) at HH stages 14-17, using a programmable microinjector (IM-300 Narishige, Japan). Right-sided labeling of the splanchnic mesoderm and the right cardinal vein was performed. Early embryos were sacrificed and analyzed 1-3 hours after injection (HH stages 14-17), and sectioned serially. Sections were stained for the myocardial marker cTnI, for Nkx2.5 which was expressed in both myocardium and mesenchyme and for Isl-1. Fluorescent labeling was found in the second heart field mesenchyme surrounding the sinus venosus and the right cardinal vein. In older stages of development (up to HH26), labeling of the developing sinus node and venous valves was found. Conclusions: Our results show that it is feasible to label the SHF and elements of the CCS in ovo, which makes it possible to trace the fate of these cells over a longer period of time. Follow-up after HH stage 26 is needed to determine whether the contributions of the SHF are not only found in the sinus node but also in the atrioventricular node, common bundle and bundle branches Future plans include earlier labeling experiments as well as contribution of the left-sided SHF and determination of the differentiation capacities of these SHF derived cells.

Vasculogenesis and the Induction of Skeletogenic Condensations in the Avian Eye

Blood vessels form via two distinct mechanisms: vasculogenesis, the formation of new blood vessels; and angiogenesis, the remodeling of preexisting blood vessels to form mature vasculature. Little research, however, focuses on the relationship between blood vessels and skeletogenic condensations, a key step in bone formation. Here, the development of the scleral ossicles in the chick begins with the induction of a neural crest-derived condensation at HH Stages 35 and 36 by overlying papillae in a 1:1 pattern. These papillae, which are epithelial thickenings of the conjunctiva, begin to form at HH Stage 30, following a distinct pattern. Nothing is currently known about their induction, or patterning. As the first papilla always forms above the ciliary artery, we mapped blood vessel development in the eye between HH Stages 28 and 36.5 using camera lucida drawings, fluorescence microscopy, and histology. Our results show that a blood vessel meshwork begins to form de novo once the ring of conjunctival papillae is complete (HH Stages 34 through 36) suggesting no direct correlation between these two events. We also observe an avascular zone beneath each conjunctival papilla, which is first visible at HH Stage 35, coinciding with the onset of induction of the skeletogenic condensations. Importantly, our findings suggest that remodeling of the vasculature and development of the avascular zones occurs at the same time as induction, but prior to the presence of the skeletogenic condensations of the intramembranous bones; this process is dissimilar to that documented for endochondral ossification in avian limb buds.

Identification of region-specific genes in the early chicken endoderm

In vertebrates, the endoderm gives rise to the epithelial lining of the digestive tract, respiratory system and endocrine organs. After gastrulation, the newly formed endoderm gradually becomes regionalized and differentiates into specific organs. To understand the molecular basis of early endoderm regionalization, which is largely unknown, it is necessary to identify novel region-specific genes as candidates potentially involved in this process. Applying an Affymetrix Array based approach we aimed for the identification of genes specifically upregulated in the foregut or mid-/hindgut endoderm at the onset of regionalization. Several genes exhibiting spatial and temporal restricted expression patterns in the developing early endoderm were identified and their expression was validated via RT-PCR and whole mount in situ hybridization. We report here the detailed gene expression patterns of two novel genes specifically associated with foregut endoderm and of eight novel genes specifically expressed in the mid-/hindgut endoderm at HH stages 10–11. Future functional analysis of these genes may help to elucidate the mechanisms involved in endoderm development and regionalization.

Expression of the zic1, zic2, zic3, and zic4 genes in early chick embryos

BackgroundThe zic genes encode a family of transcription factors with important roles during early development. Since little is known about zic gene expression in chick embryos, we have characterized the expression patterns of the zic1, zic2, zic3, and zic4 (zic1-4) genes during neurulation and somitogenesis.FindingsWe used in situ hybridization to analyze the expression patterns of the zic1-4 genes during early chick development (HH stages 7-19). The zic1-3 genes showed both overlapping and gene-specific expression patterns along the length of the dorsal neural tube and in the dorsal parts of the somites. In addition, unique expression domains of zic genes included: zic2 in the neural plate, periotic mesoderm and limb buds; zic3 in the paraxial mesoderm surrounding the neural plate, in presomitic mesoderm and in the most recently formed epithelial somites; zic2 and zic3 in developing eyes. zic4 expression was limited to dorsal fore- and midbrain regions and, unlike the expression of the zic1-3 genes, zic4 expression was not detected in the hindbrain and trunk. This was in contrast to more extensive zic4 expression in other vertebrates.ConclusionsThe zic1-3 genes were expressed in both overlapping and unique domains within the neural tube, somites and other ectoderm and mesoderm-derived structures in the future head and trunk. zic4 expression, however, was limited to dorso-anterior regions of the future brain. This is the first comprehensive study of zic1-4 gene expression in chick embryos during neurulation and somitogenesis.

Dynamic positional fate map of the primary heart-forming region

Here we show the temporal–spatial orchestration of early heart morphogenesis at cellular level resolution, in vivo, and reconcile conflicting positional fate mapping data regarding the primary heart-forming field(s). We determined the positional fates of precardiac cells using a precision electroporation approach in combination with wide-field time-lapse microscopy in the quail embryo, a warm-blooded vertebrate (HH Stages 4 through 10). Contrary to previous studies, the results demonstrate the existence of a “continuous” circle-shaped heart field that spans the midline, appearing at HH Stage 4, which then expands to form a wide arc of progenitors at HH Stages 5–7. Our time-resolved image data show that a subset of these cardiac progenitor cells do not overlap with the expression of common cardiogenic factors, Nkx-2.5 and Bmp-2, until HH Stage 10, when a tubular heart has formed, calling into question when cardiac fate is specified and by which key factors. Sub-groups and anatomical bands (cohorts) of heart precursor cells dramatically change their relative positions in a process largely driven by endodermal folding and other large-scale tissue deformations. Thus, our novel dynamic positional fate maps resolve the origin of cardiac progenitor cells in amniotes. The data also establish the concept that tissue motion contributes significantly to cellular position fate — i.e., much of the cellular displacement that occurs during assembly of a midline heart tube (HH Stage 9) is NOT due to “migration” (autonomous motility), a commonly held belief. Computational analysis of our time-resolved data lays the foundation for more precise analyses of how cardiac gene regulatory networks correlate with early heart tissue morphogenesis in birds and mammals.

The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals

BackgroundCo-ordinated cell movement is a fundamental feature of developing embryos. Massive cell movements occur during vertebrate gastrulation and during the subsequent extension of the embryonic body axis. These are controlled by cell-cell signalling and a number of pathways have been implicated. Here we use long-term video microscopy in chicken embryos to visualize the migration routes and movement behaviour of mesoderm progenitor cells as they emerge from the primitive streak (PS) between HH stages 7 and 10.ResultsWe observed distinct cell movement behaviours along the length of the streak and determined that this is position dependent with cells responding to environmental cues. The behaviour of cells was altered by exposing embryos or primitive streak explants to cell pellets expressing Wnt3a and Wnt5a, without affecting cell fates, thus implicating these ligands in the regulation of cell movement behaviour. Interestingly younger embryos were not responsive, suggesting that Wnt3a and Wnt5a are specifically involved in the generation of posterior mesoderm, consistent with existing mouse and zebrafish mutants. To investigate which downstream components are involved mutant forms of dishevelled (dsh) and prickle1 (pk1) were electroporated into the primitive streak. These had differential effects on the behaviour of mesoderm progenitors emerging from anterior or posterior regions of the streak, suggesting that multiple Wnt pathways are involved in controlling cell migration during extension of the body axis in amniote embryos.ConclusionWe suggest that the distinct behaviours of paraxial and lateral mesoderm precursors are regulated by the opposing actions of Wnt5a and Wnt3a as they leave the primitive streak in neurula stage embryos. Our data suggests that Wnt5a acts via prickle to cause migration of cells from the posterior streak. In the anterior streak, this is antagonised by Wnt3a to generate non-migratory medial mesoderm.

New empirical cell motion data from avian gastrulae reveal striking exceptions to “classical” concepts of gastrulation

An electroporation technique was recently perfected by which cells in any germ layer of a gastrulating avian embryo can be labeled with GFP (HH Stages 1–10). Combining this novel cell labeling technique with time-lapse computational imaging, we tracked the motion of cells that originated from the epiblast germ layer and discovered two interesting phenomena. First, we found that substantial numbers of labeled epiblast cells ingressed through the streak and populated the contralateral side of the embryo (HH stages 4 to 7). In some cases more than 40% of GFP-labeled cells crossed the midline axis during ingression. This observation has important consequences for understanding left-right asymmetry in early vertebrate embryos. Second, by labeling epiblast cells at HH stage 1 and monitoring their motion, we observed that some epiblast cells became mesodermal cells without ingressing through the streak. By peeling back the endoderm in the region between the area opaca and area pellucida (where epiblast cells were initially labeled with GFP), we found that most labeled cells were positioned in the mesoderm while others were associated with the ectoderm and endoderm. These empirical data establish that some epiblast cells can populate the mesoderm without gastrulating, a finding that potentially alters our concept of early vertebrate morphogenesis. Source of Support: KUMC Biomedical Research Training Program (CC), NIH R01 HL73700 (BJR), NIH R01 HL68855 (CDL).

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left–right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty, Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.

Three different fates of cells migrating from somites into the limb bud.

Cells from the ventrolateral dermomyotomal lips at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The cells give rise to myocytes and blood vascular endothelial cells (BECs) in the limb. Using vascular endothelial growth factor receptor-3 (VEGFR-3) as a marker, it has also been shown that the somites contribute to endothelial cells of lymphatic vessels in the limbs, but it is unknown where the lymphangiogenic precursors are located within the somite. In this study we used the transcription factor Prox1 as a lymphatic marker and investigated whether cells in the dorso-lateral quarter of the somite differentiate into lymphatic endothelial cells (LECs) of the limbs. To label the migrating cells, the dorso-lateral part of an epithelial brachial somite was grafted homotopically from quail into chick embryos at HH stages 13-14. The chick hosts were incubated until day 10-11 of development. The quail cell nuclei were identified with QCPN (anti-quail) antibodies. Cell differentiation was analysed by immunohistochemical staining with QH1, anti-desmin and anti-Prox1 antibodies, and by in situ hybridisation with Prox1 probes. Our results confirm that quail cell nuclei are incorporated into the myotubes of the limb muscles. Quail cells are found in the endothelium of limb blood vessels and lymphatics, predominantly the dermal lymphatics. This indicates that superficial lymphatics develop independently from the deep ones and shows that cells migrating from the lateral somitic edge into the limb buds differentiate into three cell populations: myocytes, BECs and LECs.

Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.

Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of Hox genes within a complex suggesting that multiple competing mechanisms are used to initiate their expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), we have defined two distinct groups of Hox genes based on their reciprocal sensitivity to RA or FGF during this developmental period. We found that the expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and that these same genes are refractory to RA treatment at these stages. Furthermore, we showed that the chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and that their anterior expansion is also limited to the level of the otic vesicle. Using a dominant negative form of a Xenopus Cdx gene (XcadEnR) we found that the effect of FGF treatment on 5' HoxB genes is mediated in part through the activation and function of CDX activity. Conversely, the 3' HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to RA but not FGF treatments at these stages. We demonstrated by in ovo electroporation of a dominant negative retinoid receptor construct (dnRAR) that retinoid signaling is required to initiate expression. Elevating CDX activity by ectopic expression of an activated form of a Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically activates and anteriorly expands Hoxb4 expression. In a similar manner, when ectopic expression of XcadVP16 is combined with FGF treatment, we found that Hoxb9 expression expands anteriorly into the hindbrain region. Our findings suggest a model whereby, over the window of early development we examined, all HoxB genes are actually competent to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms that axially restrict the Cdx domains of expression, serve to prevent 3' genes from responding to FGF signaling in the hindbrain. FGF may have a dual role in both modulating the accessibility of the HoxB complex along the axis and in activating the expression of Cdx genes. The position of the shift in RA or FGF responsiveness of Hox genes may be time dependent. Hence, the specific Hox genes in each of these complementary groups may vary in later stages of development or other tissues. These results highlight the key role of Cdx genes in integrating the input of multiple signaling pathways, such as FGFs and RA, in controlling initiation of Hox expression during development and the importance of understanding regulatory events/mechanisms that modulate Cdx expression.

Apoptosis Is Required for the Proper Formation of the Ventriculo-Arterial Connections

The role of apoptosis in cardiac morphogenesis has not been directly tested. Cardiomyocyte apoptosis is prevalent during the remodeling of the embryonic chicken cardiac outflow tract (OFT) in the transition from a single to a dual circulation. We tested the hypothesis that OFT cardiomyocyte apoptosis drives the shortening and rotation of the embryonic cardiac OFT and is required to achieve the mature ventriculo-arterial configuration. Chick embryos were treated with the peptide Caspase inhibitors zVAD-fmk or DEVD-cho at HH stages 15–20 (looped heart). Morphology of control and experimental embryos was assessed at HH stage 35, at which time the control hearts have developed a dual circulation. Infection of the hearts with a recombinant adenovirus expressing green fluorescent protein was used to follow the fate of the OFT cardiomyocytes. Affected embryos displayed abnormal persistence of a long infundibulum (OFT myocardial remnant) beneath the great vessels, indicating failure of OFT shortening. In some instances, the infundibulum connected both great vessels to the right ventricle in a side-by-side arrangement with transposition of the aorta, indicating a failure of rotation of the OFT, and modeling human congenital double outlet right ventricle. Defects were also observed at other sites in the heart where apoptosis is prevalent, such as in the formation of the cardiac valves and trabeculae. To more specifically target the apoptosis of the OFT cardiomyocytes, recombinant adenovirus was used to express the X-linked inhibitor of apoptosis protein in these cells. This resulted in an effect on outflow tract shortening and rotation similar to that of the peptide inhibitors, while the effects on the other cardiac structures were not observed. These results demonstrate that elimination of OFT cardiomyocytes by apoptosis is necessary for the proper formation of the ventriculo-arterial connections, and suggest apoptosis as a potential target of teratogens and genetic defects that are associated with congenital human conal heart defects.

Embryonal feather growth in the chicken.

Prenatal feather growth development in the chicken was studied in 7 body regions in HH stages 27-45, using direct measurements, specific histological and immunohistochemical methods, and scanning electron microscopy. The results from measurements of absolute length values, and, particularly, growth rate development in each HH stage revealed a distinct phase of most intensive growth in HH stage 40-41, which was preceded by feather follicle insertion and accompanied by the occurrence of alpha-keratins in barbule cells. Specific regional evaluation demonstrated that growth in the feather follicles of abdominal skin generally showed the slowest progression from absolute values and that in the feather filaments of the developing wings the most rapid progression occurred during HH stage 40-41 from growth rate values.

Regulation and Function of SF/HGF during Migration of Limb Muscle Precursor Cells in Chicken

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.

Effects of retinoic acid on chick tail bud development

The present study describes the teratogenic effects of retinoic acid (RA) on the development of the chick tail bud. Chick embryos were recovered 48 hours after treatment at HH stages 11 to 16 with various dosages of RA by subblastodermal injection. At the gross level, RA treatment resulted in varying degrees of caudal regression, scoliosis, limb malformations, and open posterior neuropores among the survivors. Histological examination of tail buds from treated embryos revealed defects which included total dysplasia of caudal structures, the presence of accessory neural tube and notochord tissue, and abnormal fusions of the notochord to the neural tube and tailgut. The incidence, severity, and location of the defects were dependent on the dose of the teratogen, and the stage of development at the time of treatment. The defects resembled those induced in previous studies by treatment with sialic acid binding lectins such as wheat germ agglutinin and limulus polyphemus lectin (Griffith and Wiley, '90b).