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doi: 10.1242/10.1242/dev.00573

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Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo

Sylvie Forlani*, Kirstie A. Lawson and Jacqueline Deschamps{dagger}

Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands



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  		Fig. 2. Schematic representation of early mouse embryogenesis and Hoxb1 and Hoxb8 expression in vivo and in explants. Under the time axis (6.5 to 8.0 days post coitus), embryonic stages (ES to LHF) are drawn, with the expression of Hoxb1 and Hoxb8 in cyan and red, respectively. Top two panels: the explants from different regions of the primitive streak are indicated above the corresponding stages. Anterior is towards the left. Posterior streak region, PSR, blue; anterior streak region, ASR, orange; median streak region, MSR, green. Lower two panels: pattern of activation of Hoxb1 and Hoxb8 in vivo and in explants. All explants contained three germ layers: the epiblast (ep), mesoderm (mes) and endoderm (end). The gastrulation stages are designated as follows: early streak (ES), early midstreak (EMS), midstreak (MS), late midstreak (LMS), late streak (LS), late streak early allantoic bud (LSEB), neural plate (NP), early headfold (EHF) and late headfold (LHF). Scale bars: 200 µm.

 


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  		Fig. 1. Hox gene activation in the mouse primitive streak. Expression of Hoxb1 (A-F) and Hoxb8 (G-J) at E7.0 to 8.0 assayed by in situ hybridisation. (A,G) Onset of expression in the proximal part of the primitive streak. (B-D,H,I) Rostral and lateral spreading of Hox expression domains towards the distal part of the streak (D,I), containing the organiser or node (n) and beyond (E,F,J). Arrowhead, rostral expression front. Lines in A and G, primitive streak. Late midstreak/late streak (A), late streak (B), late streak early bud (C), late streak early bud/neural plate (D), late neural plate (G), early headfold (E,H,I), headfold (F), late headfold (J). Anterior is towards the left, posterior towards the right. Scale bar: 100 µm.

 


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  		Fig. 3. Hoxb1 and Hoxb1-lacZ activation in cultured embryonic explants. Explants dissected at different anteroposterior levels of the primitive streak (PSR, A-D; MSR, E,F; ASR, G-J) at different stages before the onset of Hoxb1 expression in vivo, were cultured for 24 hours. Expression of Hoxb1 was assayed by in situ hybridisation after culture. A representative selection of the data is shown, and a quantitative (total numbers above bars) representation of all data obtained with Hoxb1 and Hoxb1-lacZ is given in O. Results for gene and reporter transgene were similar. (K,L) Brachyury/T and (M,N) chordin expression in ES and EMS ASR. Scale bar: 100 µm.

 


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  		Fig. 4. Hoxb8 activation in cultured embryonic explants. Posterior streak region, PSR, blue; anterior streak region, ASR, orange; median streak region, MSR, green. Hoxb8 expression in PSR (A,B), MSR (C,D) and ASR (E,F) explants cut at two different stages preceding the onset of gene expression in vivo and cultured for 24 hours. The fraction of positive explants of each type is indicated in the top right-hand corner.

 


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  		Fig. 5. Recombination with a PSR induces Hoxb1-lacZ expression in a non-expressing ASR from ES and EMS stages. Hoxb1-lacZ expression in ASR explants cut from transgenic embryos (drawn in blue) at ES or EMS stages (E6.5-6.7), cultured for 24 hours in combination with either a PSR (A) or another ASR (B) cut from wild-type embryos (E6.5, ES, EMS and MS; E7.5, EHF). Combination with a PSR explant resulted in a strong induction of transgene expression in early ASR explants. ASRs, except for the oldest (HF stage), were unable to activate Hoxb1lacZ in transgenic ES ASRs. See Table 2 for explant numbers. Scale bar: 100 µm.

 


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  		Fig. 6. HRP-labelled clones in cultured embryos. (A) Seven somite embryo labelled in the node region at the NP stage. There are seven mesoderm descendants (arrow) (not all in focus) in the second somite and a further seven lightly labelled descendants in postnodal ectoderm (star) (not all visible), which were presumably derived from a sibling still in cytoplasmic connection with the labelled cell at the time of injection. (B) Four somite embryo labelled at the anterior edge of the node region at the LS stage. Twelve labelled neurectoderm descendants are distributed in small clusters from prospective r5/r6 (arrow) towards the node. Scale bars: 100 µm.

 


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  		Fig. 7. Anteroposterior clone distribution after labelling epiblast at different stages. The upper set of figures shows results after labelling at or near the node, the lower set after labelling anterior to the node. In each set, the upper figure shows the positions of the progenitor cells projected on a sagittal section of average dimensions. The extent of the primitive streak is indicated by a curved line. A part of the sagittal section to the right of LS, LSEB and HF shows the position of progenitors initially labelled in the mesoderm layer. The positions of the clone progenitors are divided by broken lines into subregions, comprising three or four subregions from anterior to posterior in the node region and, anterior to the node, anterior axial (within 55 µm of the midline) and paraxial. Filled blue circles, progenitors with only ectoderm descendants; open blue circles, progenitors with ectoderm and mesoderm/endoderm descendants; filled red circles and squares, progenitors with only mesoderm descendants; open red circles and squares, progenitors with paraxial mesoderm and axial mesoderm or endoderm descendants; filled brown circles and squares, progenitors with only axial mesoderm descendants. The lower figures in each set show the lineal AP distribution of each clone on a schematic representation of the neural tube; the regions of the neural tube and the corresponding `segments' in the paraxial mesoderm are shown on the left. The position of the node is indicated by an asterisk. The presence of one of more labelled cells in a `segment' is indicated by a coloured line, absence of labelled cells by a broken line connecting the clone to the node. Blue, ectoderm; red, paraxial mesoderm (unless otherwise indicated); brown, axial mesoderm (notochordal plate); yellow, endoderm. The clones generated by individual progenitors can be identified as follows: for the node region, anterior precede posterior subregions and, within subregions, proximal precedes distal. Anterior towards the node, the anterior axial subregion precedes the paraxial subregion, and anterior or proximal precedes posterior or distal within each subregion. The subregional divisions are indicated by broken vertical lines on the clonal distribution scheme. Clones initiated in mesoderm follow the last epiblast subregion. Anterior clonal limits predicted by the hypothesis that Hox codes are fixed in the node region and carried further by lineage transmission are indicated by blue (neurectoderm) and red (paraxial mesoderm) arrowheads at the appropriate stages for Hoxb1 and Hoxb8. i.m., intermediate mesoderm; l.p., lateral plate mesoderm; Mes, mesencephalon; Pros, prosencephalon; r, rhombomere; Rh, rhombencephalon; S, somite; SC, spinal cord; Sm, somitomere (Meier and Tam, 1982Go); +, additional to paraxial mesoderm.

 


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  		Fig. 8. Rostral spread of Hox expression in relation to the precursors of cells that will eventually occupy the rostral expression boundaries in the neurectoderm The anteroposterior (A,P) axis is shown at different stages. The node is represented by a star, the primitive streak as an open bar. The extent of Hoxb1, Hoxb4 and Hoxb8 expression is indicated by coloured lines below the axis. The position of precursors of rhombomere 4 (R4, definitive anterior boundary of Hoxb1) and different somite (S) levels (S5 level, definitive anterior boundary of Hoxb8) in the neural axis are shown above the axis.

 


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  		Fig. 9. AP extension of hindbrain and spinal cord. (A,B) Schematic representation of the curved AP axis at the time of injection (A) and after culture (B). The position of the node is indicated by a star. For regression analysis, the position of the injected cell (x) was measured from the anterior embryonic/extra-embryonic junction (thick curved line in A). The position of the most anterior descendant (y) in the resulting clone was measured along the midline (thick curved line in B) from the boundary between the first and second somites (S1/S2). Values of y anterior to S1/S2 are negative. (C) Regression of the AP position of the most anterior descendant (y1, y2....) on progenitor position (x1, x2....). Initial stages: Square, LSEB; dots, NP; triangles, HF. Only clones with anterior axial progenitors (see Fig. 7) were used, and were suitable for this approach because there is no significant increase in length or cell mixing along the midline of prospective forebrain, midbrain and anterior hindbrain between the LS and 4S stage (K.A.L., unpublished). The values in the regression equation Y=a + bX are 137.11=–867.60 + 2.7686 (362.89).

 

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