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VOL. 71 (4), 765-782, 2005 OLD HORMONES OF THE INSULIN...

Expression of proinsulin and IGFs in chick embryo
gastrulation and neurulation

The chick embryo has been a classic vertebrate model to analyze
development from gastrulation, when the three embryo layers are
defined, to neurulation and early organogenesis. It is accessible and
indeed a beautiful artistic inspiration (Fig. 1A). Despite the lack
of a pancreatic rudiment morphologically until the third day of
incubation (E3), we found immunoreactive proinsulin/insulin in
extracts of E2 embryos by radioimmunoassay (11). The possibility of
quantifying the low amounts, as compared to pancreas, of embryonic
proinsulin mRNA was possible only by using large amounts of Poly-
A+ RNA, but confirmed the presence of transcripts in E2 chick
embryos (36). Surprisingly, even though IGF-I had been recognized as
a broadly expressed tissue growth factor, IGF-I mRNA was minimally
expressed prior to E3 in chick embryos, even when analyzed by the
highly sensitive technique of reverse transcription coupled to
polymerase chain reaction (RT-PCR) (37). Indeed, we could confirm
both by RT-PCR (31) and by in situ hybridization (Fig. 1) that during
neurulation proinsulin mRNA was broadly expressed along the
embryo whereas IGF-I was barely detectable (Fig. 1B, C) (25). In early
organogenesis, in E2.5 embryos, the expression of proinsulin mRNA
was more restricted, but still significant in developing brain, and
the two pancreatic buds (which will later be fused) were also positive
(Fig. 1D). IGF-I initial expression was mostly localized in cephalic
vesicles in E2.5 (Fig. 1E).

We were interested in defining if the protein produced by this
proinsulin transcript was unprocessed proinsulin or mature insulin
and for that purpose we developed an antiserum against the
connective C-peptide that is cleaved during processing of proinsulin
by the specific convertases. Immunofluorescence performed with this
antiserum confirmed the presence of proinsulin in distinct cells of
the three embryonic layers by E1. A few neuroepithelial cells were
the most clearly labelled (Fig. 2B). This was in contrast with the
absence of IGF-I in parallel sections (Fig. 2C), which agreed with the
previous RNA data. IGF-II was found in extracellular, likely basal
membrane, locations (Fig. 2D). With further development, in E2.5,
at mid embryo level, multiple structures expressed proinsulin,

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