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Progressively restricted expression of a new homeobox-containing gene during Xenopus laevis embryogenesis.
Su MW
,
Suzuki HR
,
Solursh M
,
Ramirez F
.
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We have isolated cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh). Northern blot and RNAase protection experiments established that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localized the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localized the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Finally, evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well.
Fig. 1. (A) Comparison of the deduced amino acid sequences of Xhox-7.1, Xhox-7.1', Quox-7 and Hox-7.1 (Hill et al.
1989; Robert et al. 1989; Takahashi and Le Douarin, 1990). The Xhox-7.1 sequence includes the additional six amino acids
encoded by clone pSU-64. Asterisks signify identity, while dots indicate gaps inserted to give best alignment. The region
comprising the sixty residues of the homeodomain is boxed. (B) Comparison of the homeodomain residues of the
Drosophila msh and the vertebrate msh-tike genes. Drosophila sequences are from Robert et al. (1989); human sequences
are from our laboratories (unpublished data) and from Ivens et al. (1990); chick sequences are from Suzuki et al.
(manuscript in preparation). Also shown is the derived consensus sequence for the msh/hoxl class of homeodomains
compared to the proposed consensus sequence for all eukaryotic homeodomains (Scott et al. 1989). Only absolutely
conserved residues are shown for the msh/hoxl consensus sequence, while the overall consensus includes highly conserved
residues that are not absolutely conserved.
Fig. 2. (A) A northern blot of total RNA hybridized first to a Xhox-7.1 probe and then to cytoskeletal gamma actin, top
and bottom respectively. Total RNA samples (10 fig in each track) are from embryos of stages 2 to 48. (N/F) denotes the
numerical stage according to Nieuwkoop and Faber (1967). Autoradiographic exposure was 4 days for Xhox-7.1 and 1 day
for actin. Size of transcripts, determined by parallel running of RNA markers, are indicated in kb. (B and C) RNAase
protection of total RNA samples (20 jig in each track) from Xenopus eggs (E) and embryos of stages 7 to 23 hybridized to
Xhox-7.1 (B) and Xhox-7.1' (C) riboprobes. Exposure of the autoradiograms was for 4 days. In each panel, the
autoradiography on the left is a shorter exposure (12 h) of the same gel showing the undigested labelled riboprobe (P).
Note that the shortening of the antisense riboprobes after RNAase treatment is due to elimination of vector sequence.
Sizes of fragments are indicated in bp. (D) Profile of Xhox-7.1 mRNA accumulation plotted after quantitation of signals
observed in the northern (A) and slot-blot hybridizations (not shown) of total RNA (5/ig) prepared at hourly intervals
from unfertilized eggs and stages 8 to 40 embryos. Values are normalized for embryo number. Developmental periods and
(N/F) stages are shown below the graph. MBT, midblastula transition; G, gastrulation; N, neurulation; TB, tailbud and
TP, tadpole stages. The dotted line denotes the period when Xhox-7.1 expression is not well defined.
Fig. 3. Expression of Xhox-7.1 at gastrula stage. On the top, cross-section at stage 11; dark-field (left) and bright-field
(right) micrographs. Note the strong signal for Xhox-7.1 in the dorsal mesodermal mantle (m). In serial cross-sections, this
distribution can be observed in the mesoderm all along the anterior-posterior axis. On the bottom, mid-sagittal section of
stage 11 embryo; dark-field (left) and bright-field (right) micrographs. The localization of Xhox-7.1 message to the dorsal
mesodermal mantle (m) is observed in this mid-sagittal section as well. A strong signal is detected along the whole
anterior-posterior axis in the chordamesoderm from the level of the dorsal lip (arrowhead) (including both the deep and
superficial layers of the mesoderm), along the dorsal side and extending around the anterior end towards the ventral
surface of the embryo. In serial parasagittal sections, localization in the anteriorchordamesoderm is more pronounced than
in this mid-sagittal section (not shown). There is also signal in cells of the yolk plug near the dorsal lip (arrow). No
significant signal was detected with the sense probe (not shown). Bar=100 microns.
Fig. 4. Expression of Xhox-7.1 at tailbud
stage. Cross sections at different levels of a
stage 21 embryo; dark-field (left) and brightfield
(right) micrographs. It is possible to
appreciate progressively later stages of
development by comparing progressively
more anterior sections (see diagram at the
bottom). (A) Cross section through the
curved tail bud. In this section, the signal is
localized to the dorsal region of the forming
neural tube (long arrow) and the dorsal
region of the neural folds (short arrow). (B)
Cross section cut at the level of the mid gut.
Note that the signal is now detected in the
neural crest cells (long arrow) and in the
dorsal-most regions of the neural tube and
lateral plate mesoderm (short arrow). (C)
Cross section cut at the level of the hind
brain. Note the intense signal in the dorsal
region of the hind brain, the adjacent
cranial neural crest cells (short arrow), as
well as neural crest adjacent to the pharynx
(long arrow) and cells in the cardiogenic
region, ventrally (curved arrow). (D)
Section through the head. Note the
localization of message in the neural crest
around the diencephalon (short arrow) and
midbrain (long arrow), as well as in the
same adjacent neural structures. No
distinctive signal was detected with the sense
probe (not shown). (E) Diagram indicating
the approximate level of the sections
illustrated. Bar=100 microns, neural fold
(n), somite (s), lateral plate (1), hind brain
(h).
Fig. 5. Expression of Xhox-7.1 at tadpole stage. Section through the head of a stage 46 embryo; dark-field (left) and
bright-field (right) micrographs. By this stage, the message is detected only at specific sites of the central nervous system,
such as cells in the dorsal region of the hindbrain (h). No significant signal was detected with the sense probe (not shown)
Bar=100 microns.
