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Download El Proceso. Bolsillo PDF. Concordant with this idea, vocal learning avian species have a relatively larger pool of cells in the telencephalic ventricular zone during development, and have a relatively elongated period of neurogenesis between hatching and sexual maturity, associated with a delayed enlargement of the telencephala compared to many other species  ,  ,  , . This extended period of cell proliferation is thought to endow altricial species, including vocal learners, with a greater capacity for cultural transmission of behavior via interactions with parents and peers.
This does not mean that all altricial species are vocal learners, but rather suggests that altricial brain development could be a precondition for evolving more complex behaviors, including culturally transmitted behaviors .
Here we asked whether this developmental difference in telencephalon enlargement between a precocial species and an altricial species is a fully cell-autonomous process or includes cell-interdependent processes. To address this question, we performed forebrain transplantation surgery between the altricial vocal learning zebra finches and the precocial vocal non-learning Japanese quail. We substituted embryonic telencephala from zebra finch donors into Japanese quail hosts from whom the same region had been removed at the neural tube stage, and harvested the chimeric embryos more than one week after surgery.
The donor and host portions of these chimeric brains successfully fused, and we found that the quail brain environment induced an accelerated enlargement of the transplanted zebra finch telencephalon with an associated increase in cell number and decrease in cell density. We propose that a partly cell-interdependent process influenced by developmental factors from outside of the forebrain contributes to the development of forebrain size differences among these different species.
All work was performed in compliance with the animal care and use guidelines of the Duke University Institutional Animal Care and Use Committee. The animal protocol was approved by the same committee Protocol A Each week, 1—2 eggs were harvested from each cage.
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Both Japanese quail eggs and zebra finch eggs were collected within 3 hours after the females laid the eggs. To perform avian in ovo surgeries, we followed a protocol for chicken-quail chimeras  with modifications described below. We incubated quail eggs at This incubation timing ensured that both quail and zebra finch embryos were at similar developmental Hamburger-Hamilton HH stages  , which we calculated by counting the number of somites Figure 1A. Under our conditions, about a hour window of time separated the more rapid embryonic development of the quail and the slower development of the zebra finch early embryos Figure 1A.
We thus performed surgeries at around the somite stage, range HH stages 8—13 Figure 1A , because the position of the neural tube was still relatively straight and blood vessels had not yet formed. Quail and zebra finch embryos were taken for surgery between HH stages 8 to 13 arrows. This surgery time window is 30—36 hours black bar for quail and 55—60 hours white bar for zebra finch eggs.
B Dorsal view of the neural tube in zebra finch donor before transplantation surgery. C Quail host before surgery. D Chimera immediately after surgery, labeled with fast green in the finch graft. The finch prosencephalon is outlined with a black line and the quail with a white line. At this stage, the anterior neural tube forms three major parts: 1 prosencephalon forebrain , 2 mesencephalon midbrain , and 3 rhombencephalon hindbrain. White arrow, injection location of the GFP plasmid; dashed white line, location for cutting out the transplanted prosencephalon; black arrow, boundary between zebra finch graft and quail host tissue after transplantation.
The zebra finch prosencephalon was dissected out and transferred into a dish of fresh sterile PBS, and any attached notochord left behind was carefully removed. This electroporation approach allowed for cell fate determination while avoiding false positive labeling, unlike that seen with more leaky lipophilic tracer labeling . We then laid the egg on its side, and carefully opened an approximately 1-cm 2 window of shell on the upper side, using a curved surgical scissor. The quail prosencephalon was excised from the host embryo using stainless steel microscalpels in ovo and removed using a glass micropipette Figure 1C.
The prosencephalon of the zebra finch donor was transferred to the host embryo and placed in the groove produced by the excision, in the normal rostro-caudal and dorso-ventral orientation Figure 1D. After transplantation, the surgery window of the host quail eggs was sealed with sterile surgical tape 3 M, USA and melted paraffin surrounding the window edge. For a sham control group, quail eggs were windowed, injected with blue food-coloring dye, and sealed as the chimera group.
We incubated the surgically treated quail and zebra finch chimeric embryos at Under these incubation conditions, normal zebra finch eggs hatch around embryonic day 13—14 and normal quail eggs around embryonic day 17— We collected all surviving embryos. At embryonic day 9, the skin surrounding the skull was removed. At embryonic days 12 and 16, the skin with feathers was removed from zebra finch heads, and in addition, the skull was removed from the quail and chimera heads.
We initially attempted to see if antibodies that distinguish neurons from glia such as NeuN could provide acceptable results for measuring the boundaries of the transplanted brain regions and for conducting cell counts. However, these types of staining were notably inferior to the techniques we detail below. This led us use techniques that allowed us to identify graft boundaries and count cells in an unambiguous manner. We found that we could distinguish zebra finch cells from quail host cells in interspecies chimeras by staining zebra finch and quail embryos with two nucleolus markers, hematoxylin and eosin HE and DAPI, and the quail cell marker antibody QCPN QCPN antibody; .
For HE staining, paraffin sections of avian embryos on Superfrost plus glass slides Fisher Scientific, USA were deparaffinized in xylene, rehydrated in a diluted ethanol series, and stained in Harris hematoxylin solution. After washing, the sections were counterstained with eosin-phloxine solution, dehydrated, delipidized and coverslipped with Permount medium Fisher Scientific, USA. For double and triple labeling of GFP with other markers, we applied sequential immunocytochemisty staining on fixed frozen sections.
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The QN antibody was kindly provided by Dr. Tanaka, Kumamoto University.
The sections were counterstained with DAPI. For measuring the optical density of QN staining, we stained both experimental and control sections at the same time with the same procedure to eliminate any experimental batch effects. All photos of QN stained sections were carefully taken under the same microscopy settings.
We used the Photoshop 7 Adobe, CA histogram function to measure QN optical density and normalized it using the background optical density without tissue on the slide. To determine brain boundaries in embryos, we used DAPI staining of chimeras and in-situ hybridizations applied to sections generated for another project, which had been hybridized with genes such as FoxP1, CoupTF2, Lxh9, and Dlx6 that define brain regional boundaries such as mesopallium, nidopallum, arcopallium, and striatum respectively; see abbreviations in Table 1 to help us confirm the brain region localizations reported here Chen et al, in preparation.
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In this study, DAPI staining of all samples provided clear morphological boundaries for brain area, and clear visualization of cell nuclei. Brain sizes were measured by systematically sampling every ten sections, estimating the total volume from the surface areas and thickness of the sections. Cell numbers and nucleus sizes were quantified stereologically by systematic random sampling using the Stereo Investigator system version 6, Microbrightfield, Burlington, VT.
Cells containing a strong DAPI signal in the nucleus with a clear edge at the nuclear envelope were counted and measured for nucleus size. Cell density was automatically calculated from sampled cell numbers and area volumes by the Stereo Investigator system. For the QN optical density analyses, if the data passed normality and equal variance tests, a parametric t-test was applied, and if not a Mann-Whitney rank sum test was applied. All statistical tests were performed using Sigma Stat 3. Zebra finches and quails are at distal ends of the neoaves phylogenetic tree, separated by an estimated divergence time of 65 million years or more .
They differ both in the duration of their incubation period and their size at birth quails have a longer incubation period and a much larger body size. Knowledge of the chronology of development in each species is a prerequisite to choosing the best stage for transplantation. Following the standard approach for early chicken embryos  ,  , we used the number of somites for each species under our incubation conditions to calculate comparative developmental stages Figure 1A.
After mastering the surgical techniques and incubation conditions, we were able to keep chimeric embryos alive up until embryonic day 16 ED16 , one day before the quail host hatching date. Our surgical-windowed control quail embryos did hatch. Under our incubation conditions, Japanese quail eggs hatched at 17 days of incubation and zebra finch eggs hatched at 13 days.
Extrapolating from the early developmental curves, ED16 in quail chronology would be similar to post-hatching day 3 for zebra finches. Thus, we were able to obtain embryos whose brains should have been at post-hatching stages according to a putative intrinsic zebra finch developmental schedule, and could therefore address questions about brain size differences in the developing embryos. We harvested chimeric embryos at ED9, 12 and 16 using the quail chronology of development and found that they exhibited many characteristics of normal body development. The heads of the zebra finch-quail forebrain chimeras successfully fused, and showed zebra finch characteristics in the anterior dorsal forehead, including the feathers, skin, eyes and beak Figure 2 ; white arrows.
Inside the skull, the zebra finch telencephalon was well attached to the rest of the host brain at its dorsal, lateral, and ventral aspects Figure 3. The grafted zebra finch retinae a derivative of the prosencephalon were also well connected onto the host optic tecta via the optic nerves projecting from the finch eyes Figure S1.
B; F vs. G , but still noticeably smaller than normal sham control quail telencephala Figure 3B vs. C; D vs. E; G vs. The remainder of the chimeric brain was morphologically similar to a normal quail brain Figure 3. Upper row, lateral views of zebra finch embryos and post hatch day 3 animal. Middle row, zebra finch-quail forebrain chimeras.
Bottom row, quail embryos. The eye, forehead, and upper beak of chimeras white arrows are derived from zebra finch graft during the surgery, whereas the bottom beak, hind head and necks of chimeras black arrows are from quail host. Dorsal A—C , ventral D—E , and lateral F—G views of whole brain morphology of each of the three groups, showing that the ZQ chimera is intact and well connected between the grafted forebrain and host brain. The zebra finch brain was left inside the thin skull, as removing it as this age is very delicate and the brain was easily destroyed by adhering to the thin skull.