Title Uloga čimbenika stanične smrti, stanične proliferacije i diferencijacije tijekom razvoja udova u čovjeka Title (english) The role of cell death, cell proliferation and differentiation factors during human limb development Author Tina Bečić Mentor Mirna Saraga-Babić (mentor)Committee member Snježana Tomić (predsjednik povjerenstva)Committee member Gordana Jurić-Lekić (član povjerenstva)Committee member Tatijana Zemunik (član povjerenstva)Granter University of Split Defense date and country 2018, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 611 - Anatomy Abstract Razvoj udova često se koristi kao model za istraživanje načina na koji genski izražaj određuje
razlike koje se između pojedinih vrsta pojavljuju tijekom normalnog i poremećenog
oblikovanja udova. Usprkos mnogobrojnim istraživanjima provedenim na oblikovanju prstiju
u pokusnih životinja, samo je nekoliko istraživanja analiziralo razvoj ljudskih udova
ostavljajući tako ovaj problem nerazjašnjenim. U ovoj je studiji razvoj ljudskih udova
analiziran u serijskim histološkim rezovima 16 ljudskih zametaka starosti od 4. do 10.
razvojnih tjedana, pri čemu su korištene imunohistokemijske metode te metode dvostruke
imunofluorescencije i elektronske mikroskopije. Statistički podaci analizirani su Mann–
Whitney, Kruskal–Wallis i Dunnovims post hoc testom.
Posebna pažnja posvećena je razvoju prstiju, naročito čimbenicima koji su uključeni u njihovo
razdvajanje i izduživanje. Prostorni i vremenski izražaj istraživanih čimbenika važnih za
kontrolu stanične proliferacije uključivali su biljega stanične proliferacije Ki-67 i
antiapoptotsku bjelančevinu Bcl-2, kao i fibroblastne čimbenike rasta FGF8 i FGF2, njihov
receptor FGFR1 te transkripcijske čimbenike MSX-1 i MSX-2. Procesi apoptoze analizirani
su primjenom čimbenika kaspaze-3, AIF, BAX, kao i čimbenika povezanih s apoptozom p19 i
RIP5. Apoptoza je dodatno dokazana TUNEL metodom.
Tijekom ranog razvoja, ljudski udovi sastojali su se od mezenhimskog središta i površinskog
ektoderma s apikalnim ektodermalnim grebenom (AEG). S napredovanjem razvoja, jaka
proliferacijska aktivnost postepeno se smanjivala u mezenhimu (od 78% na 68%) i u epitelu
(od 62% na 42%), dok je u hrskavicama prstiju proliferacija stalno bila niska (26-27%).
Apoptotske stanice pozitivne na kaspazu-3 i AIF bile su svojstvene za mezenhim i AEG
tijekom ranih faza razvoja, dok su tijekom razdvajanja prstiju bile prisutne i u mezenhimu
između prstiju (interdigitalnom mezenhimu). Intenzivni izražaj Bcl-2 uočen je u AEG,
mezenhimu neposredno ispod AEG i u hrskavičnim falangama, dok je izražaj BAX bio
svojstven područjima s apoptozom. Na ultrastrukturnoj razini, proliferirajuće stanice su imale
prepoznatljive mitotske figure, dok su apoptotske stanice pokazivale fragmentaciju jezgre.
Makrofagi su uočeni oko apoptotskih stanica.
Tijekom razvoja, pojačavao se izražaj svih fibroblastnih čimbenika rasta (osim FGF8) u
mezenhimu i AEG-u, mezenhimu neposredno ispod grebena (zona rasta) i hondrocitima
hrskavica udova. Dok je istovremeni citoplazmatski izražaj (ko-ekspresija) MSX-1 i MSX-2 nađen u epitelu i mezenhimu, p19 je pokazivao snažni citoplazmatski izražaj u neproliferirajućim
stanicama. Jezgrin izražaj Ki-67 u proliferirajućim stanicama, a djelomično i
čimbenika MSX-1 i MSX-2 uočen je u čitavoj osnovi udova. Snažni izražaj p19 i RIP5 bio je
prisutan u AEG-u i mezenhimu udova.
Rezultati naših istraživanja pokazuju da intenzivna stanična proliferacija omogućuje rast i
izduživanje osnove udova, kao i različiti rast prstiju. Dva puta stanične smrti, kaspaza-3
ovisni i AIF-ovisni put, uključeni su u kontrolu veličine AEG-a i broja stanica u podležećem
mezenhimu u najranijim razvojnim stadijima, kao i u proces odvajanja prstiju tijekom kasnijih
faza razvoja udova. Prostorni i vremenski izražaj Bcl-2 i BAX u osnovi udova ukazuje na
njihovu moguću ulogu u suzbijanju apoptoze i selektivnom poticanju rasta tijekom
oblikovanja udova. Obrazac izražaja čimbenika p19 i RIP5 ukazuje na njihovu uključenost u
kontroli staničnog starenja i smrti. Nasuprot istraživanjima na životinjama, izražaj FGFR1 u
površinskom epitelu i p19 u čitavoj osnovi ljudskih udova mogli bi biti rezultat
razlika u oblikovanju udova između različitih vrsta. Izražaj FGF2 i podređenog mu RIP5
gena, kao i transkripcijskih čimbenika MSX-1 i MSX-2 nije bio specifičan za čovjeka u
odnosu na druge vrste. Temeljem prostornog i vremenskog izražaja istraživanih čimbenika
tijekom razvoja ljudskih udova, naši rezultati upućuje na ulogu FGF i MSX gena u poticanju
stanične proliferacije, rastu udova, izduživanju i odvajanju prstiju, te dodatno na ulogu MSX-
2 u kontroli vaskulogeneze. Kaskada međusobno orkestriranih genskih izražaja, uključujući i
one analizirane u ovoj studiji, zajednički doprinosi složenom procesu razvoja udova.
Abstract (english) The limb development has been widely used as a model to investigate how gene expression
controls interspecies differences appearing in normal limb morphology and in limb
malformations. Despite numerous investigations performed on digit formation of
experimental animals, only few investigations analyzed human limb development, thus
remaining this problem largely unclear. In the present study, the development of human limbs
was analyzed in serial histological sections of 16 normal human conceptuses between the 4th
and 10th developmental weeks by using immunohistochemical and double
immunofluorescence methods, and electron microscopy. Statistical data were analyzed by
Mann–Whitney test, Kruskal–Wallis and Dunn’s post hoc test.
Special attention was paid to development of fingers, in particular to factors involved in their
separation and elongations. Spatiotemporal expression pattern of factors involved in control
of cell proliferation included proliferation marker Ki-67 and antiapoptotic marker Bcl-2, as
well as fibroblast growth factors FGF8 and FGF2, their receptor FGFR1, and transcription
factors MSX-1 and MSX-2. Processes of apoptosis were analyzed by markers to caspase-3,
AIF, BAX as well as to apoptosis associated factors19 and RIP5. Apoptosis was additionally
conformed by TUNEL method.
Initially, developing human limbs consisted of mesenchymal core and surface ectoderm with
apical ectodermal ridge (AER). During progression of development, strong proliferation
activity gradually decreased in the mesenchyme (from 78% to 68%) and in the epithelium
(from 62% to 42%), while in the differentiating finger cartilages proliferation was constantly
low (26–7%). Apoptotic caspase-3 and AIF—positive cells characterized mesenchyme and
AER at earliest stages, while during digit separation they appeared in interdigital mesenchyme
as well. Strong Bcl-2 expression was observed in AER, subridge mesenchyme and phalanges,
while BAX expression characterized limb areas undergoing apoptosis. Ultrastructurally,
proliferating cells showed mitotic figures, while apoptotic cells were characterized by nuclear
fragmentation. Macrophages were observed around the apoptotic cells.
Increasing expression of all analyzed growth factors (except FGF8) characterized both the
multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone)
and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1
and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating
cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordia. Strong
expression of factors p19 and RIP5, both in the AER and mesenchyme was observed in
human developing limbs.
Our data suggest that intense proliferation enables growth and elongation of human limb
primordia, and differential growth of digits. Both caspase-3 and AIF–dependant pathways of
cell death control the extent of AER and number of cells in the subridge mesenchyme at
earliest developmental stages, as well as process of digit separation at later stages of limb
development. Spatio-temporal co-expression of Bcl-2 and BAX indicates their role in
suppression of apoptosis and selective stimulation of growth during human limb
morphogenesis. Expression pattern of p19 and RIP5 indicates their possible involvement in
control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1
in the surface ectoderm and p19 in the whole limb primordia might reflect interspecies
differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and
transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression
pattern. Based on their spatio-temporal expression during human limb development, our study
indicates role of FGFs and MSX genes in stimulation of cell proliferation, limb outgrowth,
digit elongation and separation, and additionally MSX- 2 in control of vasculogenesis. The
cascade of orchestrated gene expressions including the analyzed developmental factors,
jointly contribute to the complex human limb development.
Keywords
Ekstremiteti -- embriologija
Ekstremiteti -- anatomija i histologija
Proliferacija stanica
Apoptoza
Keywords (english)
Extremities -- embryology
Extremities -- anatomy and histology
Cell Proliferation
Apoptosis
Language croatian URN:NBN urn:nbn:hr:171:517673 Study programme Title: Biology of Neoplasms Type of resource Text File origin Born digital Access conditions Open access Terms of use Repository MEFST Repository Created on 2023-05-12 06:58:36
